Plant cellulose synthase and promoter sequences

ABSTRACT

Provided are two plant cDNA clones that are homologs of the bacterial CelA genes that encode the catalytic subunit of cellulose synthase, derived from cotton ( Gossypium hirsutum ). Also provided are genomic promoter regions to these encoding regions to cellulose synthase. Methods for using cellulose synthase in cotton fiber and wood quality modification are also provided.

TECHNICAL FIELD

[0001] This invention relates to plant cellulose synthase cDNA encodingsequences, and their use in modifying plant phenotypes. Methods areprovided whereby the sequences can be used to control or limit theexpression of endogenous cellulose synthase.

[0002] This invention also relates to methods of using in vitroconstructed DNA transcription or expression cassettes capable ofdirecting fiber-tissue transcription of a DNA sequence of interest inplants to produce fiber cells having an altered phenotype, and tomethods of providing for or modifying various characteristics of cottonfiber. The invention is exemplified by methods of using cotton fiberpromoters for altering the phenotype of cotton fiber, and cotton fibersproduced by the method.

BACKGROUND

[0003] In spite of much effort, no one has succeeded in isolating andcharacterizing the enzyme(s) responsible for synthesis of the major cellwall polymer of plants, cellulose.

[0004] Numerous efforts have been directed toward the study of synthesisof cellulose (1,4-β-D-glucan) in higher plants. However, hampered by lowrates of activity in vitro, the cellulose synthase of plants hasresisted purification and detailed characterization (for reviews, see1,2). Aided by the discovery of cyclic-di-GMP as a specific activator,the cellulose synthase of the bacterium Acetobacter xylinum can beeasily assayed in vitro, has been purified to homogeneity, and acatalytic subunit identified (for reviews, see 2,3). Furthermore, anoperon of four genes involved in cellulose synthesis in A. xylinum hasbeen cloned (4-7).

[0005] Characterization of these genes indicates that the first gene,termed either BcsA (7) or AcsAB (6) codes for the 83 kD subunit of thecellulose synthase that binds the substrate UDP-glc and presumablycatalyzes the polymerization of glucose residues to 1,4-β-D-glucan (8).The second gene (B) of the operon is believed to function as aregulatory subunit binding cyclic-di-GMP (9) while recent-evidencesuggests that the C and D genes may code for proteins that form a poreallowing secretion of the polymer and control the pattern ofcrystallization of the resulting microfibrils (6).

[0006] Recent studies with another gram-negative bacterium,Agrobacterium tumefaciens, have also led to cloning of genes involved incellulose synthesis (10,11), although the proposed pathway of synthesisdiffers in some respects from that of A. xylinum. In A. tumefaciens, aCelA gene showing significant homology to the BcsA/AcsAB gene of A.xylinum, is proposed to transfer glc from UDP-glc to a lipid acceptor;other gene products may then build up a lipid oligosaccharide that isfinally polymerized to cellulose by the action of an endo-glucanasefunctioning in a synthetic mode. In addition, homologs of the CelA, B,and C genes have been identified in E. coli, but, as this organism isnot known to synthesize cellulose in vivo, the function of these genesis not clear (2).

[0007] These successes in bacterial systems opened the possibility thathomologs of the bacterial genes might be identified in higher plants.However, experiments in a number of laboratories utilizing the A.xylinum genes as probes for screening plant cDNA libraries have failedto identify similar plant genes. Such lack of success suggests that, ifplants do contain homologs of the bacterial genes, their overallsequence homology is not very high. Recent studies analyzing theconserved motifs common to glycosyltransferases using either UDP-glc orUDP-GlcNAc as substrate suggest that there are specific conservedregions that might be expected to be found in any plant homolog of thecatalytic subunit (referred to hereafter as CelA). In one of thesestudies, Delmer and Amor (2) identifed a motif common to many suchglycosyltransferases including the bacterial CelA proteins. Anindependent analysis (6) also concluded that this motif was highlyconserved in a group of similar glycosyltransferases.

[0008] Extending these studies further, Saxena et al. (12) presented anelegant model for the mechanism of catalysis for enzymes such ascellulose synthase that have the unique problem of synthesizingconsecutive residues that are rotated approximately rotated 180° withrespect to each other. The model invokes independent UDP-glc bindingsites and, based upon hydrophobic cluster analysis of these enzymes, theauthors concluded that 3 critical regions in all such processiveglycosyltransferases each contain a conserved aspartate (D) residue,while a fourth region contained a conserved QXXRW motif. The first Dresidue resides in the motif as previously analyzed (2,6).

[0009] In general, genetic engineering techniques have been directed tomodifying the phenotype of individual prokaryotic and eukaryotic cells,especially in culture. Plant cells have proven more intransigent thanother eukaryotic cells, due not only to a lack of suitable vectorsystems but also as a result of the different goals involved. For manyapplications, it is desirable to be able to control gene expression at aparticular stage in the growth of a plant or in a particular plant part.For this purpose, regulatory sequences are required which afford thedesired initiation of transcription in the appropriate cell types and/orat the appropriate time in the plant's development without havingserious detrimental effects on plant development and productivity. It istherefore of interest to be able to isolate sequences which can be usedto provide the desired regulation of transcription in a plant cellduring the growing cycle of the host plant.

[0010] One aspect of this interest is the ability to change thephenotype of particular cell types, such as differentiated epidermalcells that originate in fiber tissue, i.e. cotton fiber cells, so as toprovide for altered or improved aspects of the mature cell type. Cottonis a plant of great commercial significance. In addition to the use ofcotton fiber in the production of textiles, other uses of cotton includefood preparation with cotton seed oil and animal feed derived fromcotton seed husks.

[0011] A related goal involving the control of cell wall andcharacteristics would be to affect valuable secondary treecharacteristics of wood for paper forestry products. For instance, byaltering the balance of cellulose and lignin, the quality of wood forpaper production may be improved.

[0012] Finally, despite the importance of cotton as a crop, the breedingand genetic engineering of cotton fiber phenotypes has taken place at arelatively slow rate because of the absence of reliable promoters foruse in selectively effecting changes in the phenotype of the fiber. Inorder to effect the desired phenotypic changes, transcription initiationregions capable of initiating transcription in fiber cells duringdevelopment are desired. Thus, an important goal of cottonbioengineering research is the acquisition of a reliable promoter whichwould permit expression of a protein selectively in cotton fiber toaffect such qualities as fiber strength, length, color and dyability.

Relevant Literature

[0013] Cotton fiber-specific promoters are discussed in PCT publicationsWO 94/12014 and WO 95/08914, and John and Crow, Proc. Natl. Acad. Sci.USA, 89:5769-5773, 1992. cDNA clones that are preferentially expressedin cotton fiber have been isolated. One of the clones isolatedcorresponds to mRNA and protein that are highest during the late primarycell wall and early secondary cell wall synthesis stages. John and Crow,supra.

[0014] In plants, control of cytoskeletal organization is poorlyunderstood in spite of its importance for the regulation of patterns ofcell division, expansion, and subsequent deposition of secondary cellwall polymers. The cotton fiber represents an excellent system forstudying cytoskeletal organization. Cotton fibers are single cells inwhich cell elongation and secondary wall deposition can be studied asdistinct events. These fibers develop synchronously within the bollfollowing anthesis, and each fiber cell elongates for about 3 weeks,depositing a thin primary wall (Meinert and Delmer, (1984) PlantPhysiol. 59: 1088-1097; Basra and Malik, (1984) Int Rev of Cytol 89:65-113). At the time of transition to secondary wall cellulosesynthesis, the fiber cells undergo a synchronous shift in the pattern ofcortical microtubule and cell wall microfibril alignments, events whichmay be regulated upstream by the organization of actin (Seagull, (1990)Protoplasma 159: 44-59; and (1992) In: Proceedings of the Cotton FiberCellulose Conference, National Cotton Council of America, Memphis RN, pp171-192.

[0015] Agrobacterium-mediated cotton transformation is described inUmbeck, U.S. Pat. Nos. 5,004,863 and 5,159,135 and cotton transformationby particle bombardment is reported in WO 92/15675, published Sep. 17,1992. Transformation of Brassica has been described by Radke et al.(Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992)11:499-505.

[0016] Genes involved in lignin biosynthesis are described by Dwivedi,U. N., Campbell, W. H., Yu, J., Datla, R. S. S., Chiang, V. L., andPodila, G. K. (1994) “Modification of lignin biosynthesis in transgenicNicotiana through expression of an antisense O-methyltransferase genefrom Populus” Pl. Mol. Biol. 26: 61-71; and Tsai, C. J., Podila, G. K.and Chaing, V. L. (1995) “Nucleotide sequence of Populus tremuloidesgene for caffeic acid/5 hydroxyferulic acid O-methyltransferase” Pl.Physiol. 107: 1459; and also U.S. Pat. No. 5,451,514 (claiming the useof cinnamyl alcohol dehydrogenase gene in an antisense orientation suchthat the endogenous plant cinnamyl alcohol dehydrogenase gene isinhibited).

Other References Cited Throughout the Specification

[0017] 1. Gibeaut, D. M., & Carpita, N. C. (1994) FASEB J. 8, 904-915.

[0018] 2. Delmer, D. P., & Amor, Y. (1995) Plant Cell 7, 987-1000.

[0019] 3. Ross, P., Mayer, R., & Benziman, M. (1991) Microbiol. Rev. 55,35-58.

[0020] 4. Saxena, I. M., Lin, F. C., & Brown, R. M., Jr. (1990) PlantMol. Biol. 15, 673-683.

[0021] 5. Saxena, I. M., Lin, F. C., & Brown, R. M., Jr. (1992) PlantMol. Biol. 16, 947-954.

[0022] 6. Saxena, I. M., Kudlicka, K., Okuda, K., & Brown, R. M., Jr.(1994) J. Bacteriol. 176, 5735-5752.

[0023] 7. Wong, H. C., Fear, A. L., Calhoon,, R. D., Eidhinger, G. H.,Mayer, R., Amikam, D., Benziman, M., Gelfand, D. H., Meade, J. H.,Emerick, A. W., Bruner, R., Ben-Basat, B. A., & Tal, R. (1990) Proc.Natl. Acad. Sci. USA 87, 8130-8134.

[0024] 8. Lin, F.-C., Brown, R. M. Jr., Drake, R. R. Jr., & Haley, B. E.(1990) J. Biol. Chem. 265, 4782- 4784.

[0025] 9. Mayer, R., Ross, P., Winhouse, H., Amikam, D., Volman, G.,Ohana, P., Calhoon, R. D., Wong, H. C., Emerick, A. W., & Benziman, M.(1991) Proc. Natl. Acad. Sci. USA 88, 5472-5476.

[0026] 10. Matthysse, A. G., White, S., & Lightfoot, R. (1995a) J.Bacteriol. 177, 1069-1075.

[0027] 11. Matthysse, A. G., Thomas, D. O. L., & White, S. (1995b) J.Bacteriol. 177, 1076-1081.

[0028] 12. Saxena, I. M., Brown, R. M.,Jr., Fevre, M., Geremia, R. A., &Henrissat, B. (1995) J. Bacteriol. 177, 1419-1424.

[0029] 13. Meinert, M., & Delmer, D. P. (1977) Plant Physiol. 59,1088-1097.

[0030] 14. Delmer, D. P., Pear, J. R., Andrawis, A., & Stalker, D. M.(1995) Mol. Gen. Genet. 248, 43-51.

[0031] 15. Delmer, D. P., Solomon, M., & Read, S. M. (1991) PlantPhysiol. 95, 556-563.

[0032] 16. Nagai, K., & Thogersen, H. C. (1987) Methods in Enzymol. 153,461-481.

[0033] 17. Laemmli, U. K. (1970) Nature 227, 680-685.

[0034] 18. Kyte, J., & Doolittle, R. F. (1982) J. Mol. Biol. 157,105-132.

[0035] 19. Oikonomakos, N. G., Acharya, K. R., Stuart, D. I., Melpidou,A. E., McLaughlin, P. J., & Johnson, L. N. (1988) Eur. J. Biochem. 173,569-578.

[0036] 20. Maltby, D., Carpita, N. C., Montezinos, D., Kulow, C., &Delmer, D. P. (1979) Plant Physiol. 63, 1158-1164.

[0037] 21. Inoue, S. B., Takewaki, N., Takasuka, T., Mio, T., Adachi,M., Fujii, Y., Miyamoto, C., Arisawa, M., Furuichi, Y., & Watanabe, T.(1995) Eur. J. Biochem. 231, 845-854.

[0038] 22. Jacob, S. R., & Northcote, D. H. (1985) J. Cell Sci. 2(suppl.), 1-11.

[0039] 23. Delmer, D. P. (1987) Annu. Rev. Plant Physiol. 38, 259-290.

[0040] 24. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., &Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410

[0041] 25. Milligan, G., Parenti, M., & Magee, A. I. (1995) TIBS 20,183-186.

[0042] 26. Amor, Y., Haigler, C. H., Johnson, S., Wainscott, M., &Delmer, D. P. (1995) Proc. Natl. Acad. Sci. USA 92, 9353-9357.

[0043] 27. Amor, Y., Mayer, R., Benziman, M., & Delmer, D. P. (1991)Plant Cell 3, 989-995.

SUMMARY OF THE INVENTION

[0044] Two cotton genes, CelA1 and CelA2, have been shown to be highlyexpressed in developing fibers at the onset of secondary wall cellulosesynthesis. Comparisons indicate that these genes and the rice CelA geneencode polypeptides that have three regions of reasonably high homology,both in terms of primary amino acid sequence and hydropathy, withbacterial CelA proteins. The fact that these homologous stretches are inthe same sequential order as in the bacterial CelA proteins and alsocontain four sub-regions previously predicted to be critical forsubstrate binding and catalysis (12) argues that the plant genes encodetrue homologs of bacterial CelA proteins. Furthermore, the pattern ofexpression in fiber as well as our demonstration that at least one ofthese highly-conserved regions is critical for UDP-glc binding alsosupports this conclusion.

[0045] Novel DNA promoter sequences are also supplied, and methods fortheir use are described for directing transcription of a gene ofinterest in cotton fiber.

[0046] The developing cotton fiber is an excellent system for studies oncellulose synthesis as these single cells develop synchronously in theboll and, at the end of elongation, initiate the synthesis of a nearlypure cellulosic cell wall. During this transition period, synthesis ofother cell wall polymers ceases and the rate of cellulose synthesis isestimated to rise nearly 100-fold in vivo (13). In our continuingefforts to identify genes critical to this phase of fiber development,we have initiated a program sequencing randomly selected cDNA clonesderived from a library prepared from mRNA harvested from fibers at thestage in which secondary wall synthesis approaches its maximum rate(approximately 21 dpa).

[0047] We have characterized two cotton (Gossypium hirsutum) cDNA clonesand identified one rice (Oryza sativa) cDNA that are homologs of thebacterial CelA genes that encode the catalytic subunit of cellulosesynthase. Three regions in the deduced amino acid sequences of the plantCelA gene products are conserved with respect to the proteins encoded bybacterial CelA genes. Within these conserved regions are four highlyconserved subdomains previously suggested to be critical for catalysisand/or binding of the substrate UDP-glc. An overexpressed DNA segment ofthe cotton CelAl gene encodes a polypeptide fragment that spans thesedomains and effectively binds UDP-glc, while a similar fragment havingone of these domains deleted does not. The plant CelA genes show littlehomology at the amino and carboxy terminal regions and also contain twointernal insertions of sequence, one conserved and one hypervariable,that are not found in the bacterial gene sequences. Cotton celA1 andCelA2 genes are expressed at high levels during active secondary wallcellulose synthesis in the developing fiber. Genomic Southern analysesin cotton demonstrate that CelA comprises a family of approximately fourdistinct genes.

[0048] We report here the discovery of two cotton genes that showhighly-enhanced expression at the time of onset of secondary wallsynthesis in the fiber. The sequences of these two cDNA clones, termedcelA1 and CelA2, while not identical, are highly homologous to eachother and to a sequenced rice EST clone discovered in the dBESTdatabank. The deduced proteins also share significant regions ofhomology with the bacterial CelA proteins. Coupled with their high leveland specificity of expression in fiber at the time of active cellulosesynthesis, as well as the ability of an E. coli expressed fragment ofthe celA1 gene product to bind UDP-glc, these findings support theconclusion that these plant genes are true homologs of the bacterialCelA genes.

[0049] The methods of the present invention include transfecting a hostplant cell of interest with a transcription or expression cassettecomprising a cotton fiber promoter and generating a plant which is grownto produce fiber having the desired phenotype. Constructs and methods ofthe subject invention thus find use in modulation of endogenous fiberproducts, as well as production of exogenous products and in modifyingthe phenotype of fiber and fiber products. The constructs also find useas molecular probes. In particular, constructs and methods for use ingene expression in cotton embryo tissues are considered herein. By thesemethods, novel cotton plants and cotton plant parts, such as modifiedcotton fibers, may be obtained.

[0050] The sequences and constructs of this invention may also be usedto isolate related cellulose synthase genes from forest tree species,for use in transforming and modifying wood quality. As and example,lignin, an undesirable by-product of the pulping process, by be reducedby over-expressing the cellulose synthase product and divertingproduction into cellulose.

[0051] Thus, the application provides constructs and methods of userelating to modification of cell and cell wall phenotype in cotton fiberand wood products.

DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1. Northern analysis of celA1 gene in cotton tissues anddeveloping fiber. Approximately 10 μg total RNA from each tissue wasloaded per lane. Blots were prepared and probe preparation andhybridization conditions were performed as described previously (14).The entire celA1 cDNA insert was used as a probe in this experiment.Exposure time for the audoradiogram was seven hours at −70°.

[0053]FIG. 2. Cotton genomic DNA analysis for both the celA1 and CelA2cDNAs. Approximately 10-12μg of DNA was digested with the designatedrestriction enzymes and electrophoresed 0.9% agarose gels. Probepreparation and hybridization conditions were as described previously(14). The entire celA1 and CelA2 cDNAs were utilized as probes. Exposuretime for the audoradiograms was three days at −70°.

[0054]FIG. 3. Multiple alignment of deduced amino acid sequences ofplant and bacterial CelA proteins. Analyses were performed by ClustalAnalysis using the Lasergene Multalign program (DNAStar, Madison, Wis.)with gap and gap-length penalties of 10 and a PAM250 weight table.Residues are boxed and shaded when they show chemical group similarityin 4 out of 7 proteins compared. H-1, H-2, H-3 regions are indicatedwhere homology between plant and bacterial proteins is highest. Theplant proteins show two insertions that are not present in the bacterialprotein—one, P-CR, is conserved among the plant CelA genes, while asecond insertion is hypervariable (HVR) between plant genes. Thepresence of the P-CR and HVR regions led to inaccurate alignments whenthe entire proteins were compared; the optimal alignments shown herewere thus performed in five seperate blocks. Regions U-1 through U-4 arepredicted to be critical for UDP-glc binding and catalysis in bacterialCelA proteins; the predicted critical D residues and QXXRW motif areboxed and starred respectively. Potential sites of N-glycosylation areindicate by -G-.

[0055]FIG. 4. Kyte-Doolittle hydropathy plots of cotton celA1 alignedwith those of two bacterial CelA proteins. Alignments and designationsare based upon those noted in FIG. 2. The hydropathy profiles shown werecalculated using a window of 7, although a window of 19 was used forpredictions of transmembrane helices that are indicated by the arrows.

[0056]FIG. 5. An E. coli expressed GST cotton CelA-l fusion proteinbinds the containing U1 through U4 binds UDP-glc in vitro. Panel A showsa hypothetical orientation of the cotton celA1 protein in the plasmamembrane and indicates the cytoplasmic region containing the sub-domainsU-1 to U-4. GST-fusion constructs for celA1 fragments spanning theregion between the potential transmembrane helices (A through H) wereprepared as described in Materials and Methods. The purified and blottedcelA1 fusion protein fragments were tested as described in Materials andMethods for their ability to bind ³²P-UDP-glc (panel B). M refers to themolecular weight markers while CS and •U1 to the full-length and deletedGST-celA1 fusion polypeptides. The left panel shows proteins stainedwith Coomassie blue while the other three panels show representativeautoradiograms under different binding conditions as described inMaterials and Methods. Ph, BSA and Ova refer to the molecular weightstandards phosphorylase b, bovine serum albumin and ovalbuminrespectively.

[0057]FIG. 6. Nucleic acid sequences to cDNA of celA1 protein of cotton(Gossypium hirsutum).

[0058]FIG. 7. Nucleic acid sequences to cDNA of CelA2 protein of cotton(Gossypium hirsutum), including approximately the last 3′ two-thirds ofthe encoding region.

[0059]FIG. 8. Genomic nucleic acid sequences of celA1 protein of cotton(Gossypium hirsutum), including approximately 900 bases of the promoterregion 5′ to the encoding sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0060] In accordance with the subject invention, novel constructs andmethods are described, which may be used provide for transcription of anucleotide sequence of interest in cells of a plant host, preferentiallyin cotton fiber cells to produce cotton fiber having an altered colorphenotype.

[0061] Cotton fiber is a differentiated single epidermal cell of theouter integument of the ovule. It has four distinct growth phases;initiation, elongation (primary cell wall synthesis), secondary cellwall synthesis, and maturation. Initiation of fiber development appearsto be triggered by hormones. The primary cell wall is laid down duringthe elongation phase, lasting up to 25 days postanthesis (DPA).Synthesis of the secondary wall commences prior to the cessation of theelongation phase and continues to approximately 40 DPA, forming a wallof almost pure cellulose.

[0062] The constructs for use in such cells may include several forms,depending upon the intended use of the construct. Thus, the constructsinclude vectors, transcriptional cassettes, expression cassettes andplasmids. The transcriptional and translational initiation region (alsosometimes referred to as a “promoter,”), preferably comprises atranscriptional initiation regulatory region and a translationalinitiation regulatory region of untranslated 5′ sequences, “ribosomebinding sites,” responsible for binding mRNA to ribosomes andtranslational initiation. It is preferred that all of thetranscriptional and translational functional elements of the initiationcontrol region are derived from or obtainable from the same gene. Insome embodiments, the promoter will be modified by the addition ofsequences, such as enhancers, or deletions of nonessential and/orundesired sequences. By “obtainable” is intended a promoter having a DNAsequence sufficiently similar to that of a native promoter to providefor the desired specificity of transcription of a DNA sequence ofinterest. It includes natural and synthetic sequences as well assequences which may be a combination of synthetic and natural sequences.

[0063] Cotton fiber transcriptional initiation regions of cellulosesynthase are used in cotton fiber modification.

[0064] A transcriptional cassette for transcription of a nucleotidesequence of interest in cotton fiber will include in the direction oftranscription, the cotton fiber transcriptional initiation region, a DNAsequence of interest, and a transcriptional termination regionfunctional in the plant cell. When the cassette provides for thetranscription and translation of a DNA sequence of interest it isconsidered an expression cassette. One or more introns may be also bepresent.

[0065] Other sequences may also be present, including those encodingtransit peptides and secretory leader sequences as desired.

[0066] Downstream from, and under the regulatory control of, thecellulose synthase transcriptional/translational initiation controlregion is a nucleotide sequence of interest which provides formodification of the phenotype of fiber. The nucleotide sequence may beany open reading frame encoding a polypeptide of interest, for example,an enzyme, or a sequence complementary to a genomic sequence, where thegenomic sequence may be an open reading frame, an intron, a noncodingleader sequence, or any other sequence where the complementary sequenceinhibits transcription, messenger RNA processing, for example, splicing,or translation. The nucleotide sequences of this invention may besynthetic, naturally derived, or combinations thereof. Depending uponthe nature of the DNA sequence of interest, it may be desirable tosynthesize the sequence with plant preferred codons. The plant preferredcodons may be determined from the codons of highest frequency in theproteins expressed in the largest amount in the particular plant speciesof interest. Phenotypic modification can be achieved by modulatingproduction either of an endogenous transcription or translation product,for example as to the amount, relative distribution, or the like, or anexogenous transcription or translation product, for example to providefor a novel function or products in a transgenic host cell or tissue. Ofparticular interest are DNA sequences encoding expression productsassociated with the development of plant fiber, including genes involvedin metabolism of cytokinins, auxins, ethylene, abscissic acid, and thelike. Methods and compositions for modulating cytokinin expression aredescribed in U.S. Pat. No. 5,177,307, which disclosure is herebyincorporated by reference. Alternatively, various genes, from sourcesincluding other eukaryotic or prokaryotic cells, including bacteria,such as those from Agrobacterium tumefaciens T-DNA auxin and cytokininbiosynthetic gene products, for example, and mammals, for exampleinterferons, may be used.

[0067] Alternatively, the present invention provides the sequences tocotton cellulose synthase, which can be expressed, or down regulated byantisense or co-suppression with its own, or other cotton or other fiberpromoters to modify fiber phenotyp.

[0068] In cotton, primary wall hemicellulose synthesis ceases assecondary wall synthesis initiates in the fiber, and there are only twopossible β-glucans synthesized in fibers at the time these genes arehighly-expressed; callose and cellulose (20). The following datastrongly argue against the plant CelA genes coding for callosesynthase: 1) callose synthase binds UDP-glc and is activated in aCa²⁺-dependent manner (2), while the celA1 polypeptide fragmentcontaining the UDP-glc binding site preferentially binds UDP-glc in aMg²⁺-dependent manner, similar to bacterial cellulose synthase (9); 2)the timing of synthesis of callose in vivo in developing cotton fiber(20) does not match the expression of the cotton CelA genes (FIG. 1); 3)comparison of the CelA gene sequences with those of suspected1,3β-glucan synthase genes from yeast (21) indicated no significanthomology.

[0069] It is still possibille that the CelA protein might encode bothactivities, as hypothesized some years ago (22-23), and the plant CelAsmight be responsible for direct polymerization of glucan from UDP-glc asproposed for A. xylinum, although they may catalyze synthesis of alipid-glc precursor as proposed for the CelA protein of A. tumefaciens.

[0070] In addition to their similarities, the plant CelA genes showseveral very interesting divergences from their bacterial ancestors, andthese may account for the previous lack of success in using bacterialprobes to detect these cDNA clones. However, a BLAST search of proteindata banks (24) using the entire protein sequence of cotton celA1 alwaysshows highest homology with the bacterial cellulose synthases. Ofparticular interest is the insertion of two unique, plant-specificregions designated P-CR and HVR. These regions are clearly not artifactsof cloning as they are observed in both cotton genes as well as the riceCelA gene. The three plant proteins show a high degree of amino acidhomology to each other throughout most of their length, diverging onlyat the N- and C-terminal ends and the very interesting HVR region. It istempting to speculate that the HVR region may confer some specificity offunction; the highly-charged and cysteine rich nature of the firstportion of HVR could make this region a potential candidate forinteraction with specific regulatory proteins, for cytoskeletalelements, or for redox regulation. In addition, we note the presence ofseveral cysteine residues near the N- and C-terminal regions of theprotein that might serve as substrates for palmytolylation and alsoserve to help anchor the protein in the membrane (25).

[0071] In summary, the finding of these plant CelA homologs potentiallyopens up an exciting chapter in research on cellulose synthesis inhigher plants. Their finding is of particular significance sincebiochemical approaches to identification of plant cellulose synthasehave proven exceedingly difficult. One obvious challenge will be to gaindefinitive proof that these genes are truely functional in cellulosesynthesisin vivo. Other promising goals will be to identify othercomponents of a complex that might interact with CelA, such as thatproposed for sucrose synthase (26), and/or a regulatory subunit thatbinds cyclic-di-GMP (9,27) or other glycosyltransferases (10,11).

[0072] Transcriptional cassettes may be used when the transcription ofan anti-sense sequence is desired. When the expression of a polypeptideis desired, expression cassettes providing for transcription andtranslation of the DNA sequence of interest will be used. Variouschanges are of interest; these changes may include modulation (increaseor decrease) of formation of particular saccharides, hormones, enzymes,or other biological parameters. These also include modifying thecomposition of the final fiber that is changing the ratio and/or amountsof water, solids, fiber or sugars. Other phenotypic properties ofinterest for modification include response to stress, organisms,herbicides, brushing, growth regulators, and the like. These results canbe achieved by providing for reduction of expression of one or moreendogenous products, particularly an enzyme or cofactor, either byproducing a transcription product which is complementary (anti-sense) tothe transcription product of a native gene, so as to inhibit thematuration and/or expression of the transcription product, or byproviding for expression of a gene, either endogenous or exogenous, tobe associated with the development of a plant fiber.

[0073] The termination region which is employed in the expressioncassette will be primarily one of convenience, since the terminationregions appear to be relatively interchangeable. The termination regionmay be native with the transcriptional initiation region, may be nativewith the DNA sequence of interest, may be derived from another source.The termination region may be naturally occurring, or wholly orpartially synthetic. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. In some embodiments, it may bedesired to use the 3′ termination region native to the cotton fibertranscription initiation region used in a particular construct.

[0074] As described herein, in some instances additional nucleotidesequences will be present in the constructs to provide for targeting ofa particular gene product to specific cellular locations.

[0075] Similarly, other constitutive promoters may also be useful incertain applications, for example the mas, Mac or DoubleMac, promotersdescribed in U.S. Pat. No. 5,106,739 and by Comai et al., Plant Mol.Biol. (1990) 15:373-381). When plants comprising multiple geneconstructs are desired, the plants may be obtained by co-transformationwith both constructs, or by transformation with individual constructsfollowed by plant breeding methods to obtain plants expressing both ofthe desired genes.

[0076] A variety of techniques are available and known to those skilledin the art for introduction of constructs into a plant cell host. Thesetechniques include transfection with DNA employing A. tumefaciens or A.rhizogenes as the transfecting agent, protoplast fusion, injection,electroporation, particle acceleration, etc. For transformation withAgrobacterium, plasmids can be prepared in E. coli which contain DNAhomologous with the Ti-plasmid, particularly T-DNA. The plasmid may ormay not be capable of replication in Agrobacterium, that is, it may ormay not have a broad spectrum prokaryotic replication system such asdoes, for example, pRK290, depending in part upon whether thetranscription cassette is to be integrated into the Ti-plasmid or to beretained on an independent plasmid. The Agrobacterium host will containa plasmid having the vir genes necessary for transfer of the T-DNA tothe plant cell and may or may not have the complete T-DNA. At least theright border and frequently both the right and left borders of the T-DNAof the Ti- or Ri-plasmids will be joined as flanking regions to thetranscription construct. The use of T-DNA for transformation of plantcells has received extensive study and is amply described in EPA SerialNo. 120,516, Hoekema, In: The Binary Plant Vector SystemOffset-drukkerij Kanters B. V., Alblasserdam, 1985, Chapter V, Knauf, etal., Genetic Analysis of Host Range Expression by Agrobacterium, In:Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed.,Springer-Verlag, NY, 1983, p. 245, and An, et al., EMBO J. (1985)4:277-284.

[0077] For infection, particle acceleration and electroporation, adisarmed Ti-plasmid lacking particularly the tumor genes found in theT-DNA region) may be introduced into the plant cell. By means of ahelper plasmid, the construct may be transferred to the A. tumefaciensand the resulting transfected organism used for transfecting a plantcell; explants may be cultivated with transformed A. tumefaciens or A.rhizogenes to allow for transfer of the transcription cassette to theplant cells. Alternatively, to enhance integration into the plantgenome, terminal repeats of transposons may be used as borders inconjunction with a transposase. In this situation, expression of thetransposase should be inducible, so that once the transcriptionconstruct is integrated into the genome, it should be relatively stablyintegrated. Transgenic plant cells are then placed in an appropriateselective medium for selection of transgenic cells which are then grownto callus, shoots grown and plantlets generated from the shoot bygrowing in rooting medium.

[0078] To confirm the presence of the transgenes in transgenic cells andplants, a Southern blot analysis can be performed using methods known tothose skilled in the art. Expression products of the transgenes can bedetected in any of a variety of ways, depending upon the nature of theproduct, and include immune assay, enzyme assay or visual inspection,for example to detect pigment formation in the appropriate plant part orcells. Once transgenic plants have been obtained, they may be grown toproduce fiber having the desired phenotype. The fibers may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants having the desired characteristics. The termstransgenic plants and transgenic cells include plants and cells derivedfrom either transgenic plants or transgenic cells.

[0079] The various sequences provided herein may be used as molecularprobes for the isolation of other sequences which may be useful in thepresent invention, for example, to obtain related transcriptionalinitiation regions from the same or different plant sources. Relatedtranscriptional initiation regions obtainable from the sequencesprovided in this invention will show at least about 60% homology, andmore preferred regions will demonstrate an even greater percentage ofhomology with the probes.

[0080] Of particular importance is the ability to obtain relatedtranscription initiation control regions having the timing and tissueparameters described herein. Thus, by employing the techniques describedin this application, and other techniques known in the art (such asManiatis, et al., Molecular Cloning,- A Laboratory Manual (Cold SpringHarbor, N.Y.) 1982), other encoding regions or transcription initiationregions of cellulose synthase as described in this invention may bedetermined. The constructs can also be used in conjunction with plantregeneration systems to obtain plant cells and plants; thus, theconstructs may be used to modify the phenotype of fiber cells, toprovide cotton fibers which are colored as the result of geneticengineering to heretofor unavailable hues and/or intensities.

[0081] Various varieties and lines of cotton may find use in thedescribed methods. Cultivated cotton species include Gossypium hirsutumand G. babadense (extra-long stable, or Pima cotton), which evolved inthe New World, and the Old World crops G. herbaceum and G. arboreum.

[0082] By using encoding sequences to enzymes which control wood qualityand wood product characteristics, i.e., cellulose synthase andO-methyltransferase (a key enzyme in lignin biosynthesis) the relativesynthesis of cellulose and lignin by plants may be controlled.Transformation of the plant genome with a recombinant gene constructwhich contains the gene specifying an enzyme critical to the synthesisof cellulose or lignin or a lignin precursor, in either a sense or in anantisense orientation. If an antisense orientation, the gene willtranscribed so mRNA having a sequence complementary to the equivalentmRNA transcribed from the endogenous gene is expressed, leading tosuppression of the synthesis of lignin or cellulose.

[0083] If the recombinant gene has the lignin enzyme gene in normal, or“sense” orientation, increased production of the enzyme may occur whenthe insert is the full length DNA but suppression may occur if only apartial sequence is employed.

[0084] Furthermore, the expression of one may be increased in thismanner while the other is reduced. For instance, the production ofcellulose may by increased through the overexpression of cellulosesynthase, while lignin production is reduced. By thus reducing therelative lignin content, the quality of wood for paper production wouldbe improved.

EXAMPLES

[0085] The following examples are offered by way of illustration and notby limitation.

Example 1 cDNA Libraries

[0086] An unamplified cDNA library was used to prepare the LambdaUni-Zap vector (Stratagene, LaJolla, Calif.) using cDNA derived frompolyA+ mRNA prepared from fibers of Gossypium hirsutum Acala SJ-2harvested at 21 DPA, the time at which secondary wall cellulosesynthesis is approaching a maximal rate (13). Approximately 250 plaqueswere randomly selected from the cDNA library, phages purified andplasmids excised from the phage vector and transformed.

[0087] The resulting clones/inserts were size screened on 0.8% agarosegels (DNA inserts below 600 bp were excluded).

Example 2 Isolation and Sequencing of cDNA Clones

[0088] Plasmid DNA inserts were randomly sequenced using an AppliedBiosystems (Foster City, Calif.) Model 373A DNA sequencer. A search ofthe GenBank EST databank revealed that there were at least 23 rice and 8Arabidopsis EST clones that contain sequences similar to the cottoncelA1 DNA sequence. EST clone S14965 was obtained from Y. Nagamura (RiceGenome Research Program, Tsukuba). A series of deletion mutants weregenerated and used for DNA sequencing analysis at the Weizmann Instituteof Science (Rehovot).

Example 3 Northern and Southern Analyses

[0089] Cotton plants (G. hirsutum cv. Coker 130) were grown in thegreenhouse and tissues harvested at the appropriate times indicated andfrozen in liquid N₂. Total cotton RNA and cotton genomic DNA wasprepared and subjected to Northern and Southern analyses as describedpreviously (14).

Example 4 UDP-Glc Binding Studies

[0090] To construct a GST-celA1 protein fusion, a 1.6 kb DNA celA1 DNAfragment containing a putative cytoplasmic domain between the second andthird transmembrane helices was PCR amplified with the primersATTGAATTCCTGGGTGTTGGATCAGTT and ATTCTCGAGTGGAAGGGATTGAAA in a reactioncontaining 1 ng plasmid DNA (clone 213) as template. The amplifiedfragment was unidirectionally cloned into the EcoRI and XhoI sites ofthe GST expression vector pGEX4T-3 (Pharmacia), generating a fusionprotein GST-CS containing the amino acids Ser215 to Leu759 of the cottoncelA1 protein. Two celA1 gene internal PstI sites within the plasmidpGST-CS were used to generate the deletion mutant pGST-CSΔU1, whichlacks 196 amino acids (and the U1 binding region) from Val252 to Ala447.

[0091] For the UDGP binding assays, α-³²P-labeled UDP-glc was preparedas described (15). The two fusion proteins GST-CS and GST-CS•U1 wereexpressed in E. coli and purified from inclusion bodies (16). Proteinswere suspended in sample buffer, heated to 100° C. for 5 min andapproximately 50 ng of the two fusion protein products and molecularweight standards (Bio-Rad) subjected to SDS-PAGE using 4.5% and 7.5%acrylamide in the stacking and separating gels, respectively (17). Afterelectrophoresis, protein transfer to nitrocellulose filters was carriedout in transfer buffer (25 mM Tris, 192 mM glycine and 20% (v/v)methanol). The filter was briefly rinsed in deionized H₂O and incubatedin PBS buffer for 15 min, then stained with Ponceau-S in PBS buffer.After washing in deionized H₂O, protein was further renatured on thefilter by incubation in PBS buffer for 30 min and used directly forbinding assays. All binding buffers contained 50 mM HEPES/KOH (pH 7.3),50 mM NaCl and 1 mMDTT. In addition, binding buffers contained either 5mM MgCl2 and 5 mM EGTA (Buffer Mg/EGTA), 5 mM EDTA (Buffer EDTA) or 1 mMCaCl2 and 2 mM cellobiose (Buffer Ca/CB). Binding reaction was carriedout in 7 ml containing ³²P-labeled UDP-glc (1×10⁷ cpm) at roomtemperature for 3 hours with constant shaking. Filters were washedseparately three times in 20 ml washing buffer consisting of 50 mMHEPES/KOH (pH 7.3) and 50 mM NaCl for 5 min each, briefly dried andanalyzed on a Bio-imaging analyzer BAS1000 (Fugi).

Example 5 Identification, Differential Expression and Genomic Analysisof Cotton CelA Genes

[0092] During the course of screening and sequencing random cDNA clonesfrom a cotton fiber specific cDNA library prepared from RNA collectedapproximately 21 dpa, it was discovered that two cDNA clones thatinitially exhibited small blocks of amino acid homology to the proteinsencoded by the bacterial CelA genes. Clone 213 appeared to befull-length cDNA while another distinct clone, 207, appeared to be apartial clone relative to the length of 213. These two clones werepartially homologous at the nucleotide and amino acid levels anddesignated celA1 and CelA2 respectively.

[0093] These clones were then utilized as probes for Northern blotanalysis to determine their differential expression in cotton tissuesand developing cotton fiber. FIG. 1 indicates the expression pattern forthe celA1 gene. The celA1 gene encodes a mRNA of approximately 3.2 kb inlength and is expressed at extremely high levels in developing fiber,beginning at approximately 17 dpa, the time at which secondary wallcellulose synthesis is initiated(13). The gene is also expressed at lowlevels in all other cotton tissues, most notably in root, flower anddeveloping seeds. Since regions of these genes are somewhat homologousat the nucleotide level, gene specific probes were designed (using thehypervariable regions described in FIG. 3) to distinguish the specificexpression patterns of celA1 and CelA2. These gene specific probesgenerated expression patterns (data not shown) for the two genesidentical to that shown in FIG. 1, except that a very low mRNA level wasalso detected in the primary wall phase of fiber development (5-14 dpa)for the CelA2 gene when the blots were overexposed. The CelA2 genespecific probe also encoded a 3.2 kb mRNA, analogous in size to the mRNAspecified by the gene for celA1. Messenger RNAs for both genes exhibit acharacteristic degradation pattern similar to other mRNAs specificallyexpressed late in fiber development (J. Pear, unpublished observations)and this degradation is not a result of the integrity of the mRNApreparations (14). We estimate that both cotton CelA genes are expressedin developing fiber approximately 500 times their level of expression inother cotton tissues and that they constitute approximately 1-2% of the24 dpa fiber mRNA.

[0094] In order to estimate the number of CelA genes in the cottongenome, Southern analysis was performed utilizing both CelA cDNAsindependently as probes (FIG. 2). Although the two cotton genes arefairly non-homologous at the nucleotide level over their entire length,there are regions of homology (the H1, H2 and H3 regions describedbelow) and it was thought these regions could be useful in identifyingother cotton CelA genes. FIG. 2 indicates that the celA1 cDNA probe willhybridize, albeit weakly, to the CelA2 genomic equivalent and viseversa. The HindIII pattern for both genes and cDNA probes isparticularly discriminating. There are also a number of other weaklyhybridzing bands in these digests and from these data we estimate thatthe cotton CelA genes constitute a small family of approximately fourgenes. Homology of Plant and Bacterial CelA Gene Products.

[0095] In addition to the two similar cotton CelA genes, a homologouscDNA clone was discovered in the dBest databank* of rice and ArabidopsisESTs. Accession No. D48636, the rice clone having the longest insert wasobtained and sequenced, and the homology comparisons with bacterialproteins reported here also include results with the rice CelA. FIG. 3shows the results of a multiple alignment of the deduced amino acidsequences from the three plant CelA genes and four bacterial CelA genesfrom A. xylinum (AcsAB and BcsA), E. coli , and A. tumefaciens. FIG. 4shows hydropathy plots (18) of cotton celA1 similarly aligned with twobacterial CelA proteins and serves as a more general summary of theoverall homologies.

[0096] Of the plant genes, only the cotton celA1 appears to be afull-length clone of 3.2 kb exhibiting an open reading frame that couldpotentially code for a polypeptide of 109,586 kD, a pI of 6.4, and fourpotential sites of N-glycosylation. Comparison of the N-terminal regionof cotton celA1 with bacterial genes indicates that the plant proteinhas an extended N-terminal similar in length and hydropathy profile, butwith only poor amino acid sequence homology to the A. tumefaciens CelAprotein. In general, sequence homology of plant and bacterial genes inboth the N-terminal and C-terminal regions is poor. However, althoughoverall similarity comparing plant to bacterial proteins is less than25%, three homologous regions were identified, called H-1, H-2, and H-3,where the sequence similarity rises to 50-60% at the amino acid level.Interspersed between these regions of homology are two plant-specificregions not found at all in the bacterial proteins. Sequences in thefirst of these insertions are highly conserved in the plant genes(P-CR), while the second interspersed region seems to be a hypervariableregions (HVR) for there is considerable sequence divergence among theplant proteins analyzed.

[0097] None of the plant or bacterial CelA proteins contains obvioussignal sequences even though they are presumably transmembrane proteins(4). However, the overall profiles suggest two potential transmembranehelices in the N-terminal and six in the C-terminal region of the cottoncelA1 that could anchor the protein in the membrane (see arrows FIG. 3and also panel A of FIG. 5). The amino acid sequence positions for thesepredicted transmembrane helices are: A (169-187), B (200-218), C(759-777), D (783-801), E (819-837), F (870-888), G (903-921), H(933-951). The central portions of the proteins are more hydrophilic andare predicted to reside in the cytoplasm and contain the site(s) ofcatalysis. More detailed inspection of these hydrophilic stretchesreveals four particularly conserved sub-regions (marked U-1 through U-4on FIGS. 3-4) that contain the conserved asp (D) residues (in U-1-3) andthe motif QXXRW (in U-4) that have been proposed (12) to be involved insubstrate binding and/or catalysis .

[0098] Binding of UDP-glucose. Further evidence that the proteinsencoded by these plant genes are CelA homologs comes from ourdemonstration that a DNA segment encoding the central region of thecotton celA1 protein, over-expressed in E. coli, binds UDP-glc. Wesubcloned a 1.6 kb fragment of the cotton celA1 clone to create a hybridgene that encodes GST fused to the celA1 sequence encoding amino acidresidues 215-759 of the celA1 protein (FIG. 5a). This region spans U-1through U-4 that are suspected to be critical for UDP-glc binding. As acontrol, another GST fusion was created using a 1.0 kb PstI fragmentthat had the U-1 region deleted and might not be predicted to bindUDP-glc. The fusion proteins were overexpressed in E. coli purifed, andshown to have the predicted sizes of approximately 87 and 64 kD,respectively (FIG. 5b). The purified proteins were then subjected toSDS-PAGE, and blotted to nitrocellulose. Blotted proteins wererenatured, and incubated with ³²P-UDP-glc in order to test for binding(FIG. 5b). As predicted, the 87 kD GST-celA1 fusion does indeed bindUDP-glc in a Mg²⁺ dependent manner, while the shorter fusion with theU-1 domain deleted did not show any binding (Although not observed inthe experiment shown, in some experiments very weak labeling in thepresence of Ca²⁺ could be observed). As further controls, note that themolecular weight standards BSA and ovalbumin, proteins lacking UDP-glcbinding sites, show no interaction with UDP-glc, while phosphorylase b,an enzyme inhibited by UDP-glc (19), binds this substrate.

[0099]FIG. 6 provides the encoding sequence to the cDNA to celAl (startATG at ˜base 179), while FIG. 7 provides the encoding sequence to theapproximately two-thirds 3′ of the cDNA to celA2.

Example 6 Genomic DNA

[0100] cDNA for the cellulose synthase clones was used to probe forgenomic clones. For both, full length genomic DNA was obtained from alibrary made using the lambda dash 2 vector from Stratagene™, which wasused to construct a genomic DNA library from cotton variety Coker 130(Gossypium hirsutum cv. coker 130), using DNA obtained from germinatingseedlings.

[0101] The cotton genomic library was probed with a cellulose synthaseprobe and genomic phage candidates were identified and purified. FIG. 8provides an approximately 1 kb sequence of the cellulose synthasepromoter region which is immediately 5′ to the celA1 encoding region.The start of the cellulose synthase enzyme encoding region is at the ATGat base number 954.

Example 7 Cotton Transformation Explant Preparation

[0102] Promoter constructs comprising the cellulose synthase promotersequences of celAl can be cotton prepared. Coker 315 seeds are surfacedisinfected by placing in 50% Clorox (2.5% sodium hypochlorite solution)for 20 minutes and rinsing 3 times in sterile distilled water. Followingsurface sterilization, seeds are germinated in 25×150 sterile tubescontaining 25 mls ½×MS salts: ½×B5 vitamins: 1.5% glucose: 0.3% gelrite.Seedlings are germinated in the dark at 28° C. for 7 days. On theseventh day seedlings are placed in the light at 28±2° C.

Cocultivation and Plant Regeneration

[0103] Single colonies of A. tumefaciens strain 2760 containing binaryplasmids pCGN2917 and pCGN2926 are transferred to 5 ml of MG/L broth andgrown overnight at 30° C. Bacteria cultures are diluted to 1×108cells/ml with MG/L just prior to cocultivation. Hypocotyls are excisedfrom eight day old seedlings, cut into 0.5-0.7 cm sections and placedonto tobacco feeder plates (Horsch et al. 1985). Feeder plates areprepared one day before use by plating 1.0 ml tobacco suspension cultureonto a petri plate containing Callus Initiation Medium CIM withoutantibiotics (MS salts: B5 vitamins: 3% glucose: 0.1 mg/L 2,4-D: 0.1 mg/Lkinetin: 0.3% gelrite, pH adjusted to 5.8 prior to autoclaving). Asterile filter paper disc (Whatman #1) was placed on top of the feedercells prior to use. After all sections are prepared, each section wasdipped into an A. tumefaciens culture, blotted on sterile paper towelsand returned to the tobacco feeder plates.

[0104] Following two days of cocultivation on the feeder plates,hypocotyl sections are placed on fresh Callus Initiation Mediumcontaining 75 mg/L kanamycin and 500 mg/L carbenicillin. Tissue isincubated at 28±2° C., 30uE 16:8 light:dark period for 4 weeks. At fourweeks the entire explant is transferred to fresh callus initiationmedium containing antibiotics. After two weeks on the second pass, thecallus is removed from the explants and split between Callus InitiationMedium and Regeneration Medium (MS salts: 40 mM KNO₃. 10 mM NH4Cl:B5vitamins:3% glucose:0.3% gelrite:400 mg/L carb:75 mg/L kanamycin).

[0105] Embryogenic callus is identified 2-6 months following initiationand was subcultured onto fresh regeneration medium. Embryos are selectedfor germination, placed in static liquid Embryo Pulsing Medium (Stewartand Hsu medium: 0.01 mg/l NAA: 0.01 mg/L kinetin: 0.2 mg/L GA3) andincubated overnight at 30° C. The embryos are blotted on paper towelsand placed into Magenta boxes containing 40 mls of Stewart and Hsumedium solidified with Gelrite. Germinating embryos are maintained at28±2° C. 50 uE m²s ¹16:8 photoperiod. Rooted plantlets are transferredto soil and established in the greenhouse.

[0106] Cotton growth conditions in growth chambers are as follows: 16hour photoperiod, temperature of approximately 80-85°, light intensityof approximately 500 μEinsteins. Cotton growth conditions in greenhousesare as follows: 14-16 hour photoperiod with light intensity of at least400 μEinsteins, day temperature 90-95° F., night temperature 70-75° F.,relative humidity to approximately 80%.

Plant Analysis

[0107] Flowers from greenhouse grown T1 plants are tagged at anthesis inthe greenhouse. Squares (cotton flower buds), flowers, bolls etc. areharvested from these plants at various stages of development and assayedfor observable phenotype or tested for enzyme activity.

Example 7 Transformation of Tree Species

[0108] Numerous methods are known to the art for transforming foresttree species, for example U.S. Pat. No. 5,654,190 discloses a processfor producing transgenic plant belonging to the genus Populus, thesection Leuce.

[0109] The above results demonstrate how the cellulose synthase cDNA maybe used to alter the phenotype of a transgenic plant cell, and how thepromoter may be used to modify transgenic cotton fiber cells.

[0110] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application are specifically and individuallyindicated to be incorporated by reference.

[0111] Although the foregoing invention has been described in somedetail, by way of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art that certain changes and modifications may be made thereto,without departing from the spirit or scope of the appended claims.

1 12 1 3328 DNA Artificial Sequence Synthetic Oligonucleotide 1cgaaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc 60tagaactagt ggatcccccg ggctgcagga attcggcacg agggttagca tattgtttgt 120agcattgggt ttttttctca aggaagaaga aggagaaaga taagtacttt ttttgagaat 180gatggaatct ggggttcctg tttgccacac ttgtggtgaa catgttgggt tgaatgttaa 240tggtgaacct tttgtggctt gccatgaatg taatttccct atttgtaaga gttgttttga 300gtatgatctt aaggaaggac gaaaagcttg cttgcgttgt ggtagtccat atgatgaaaa 360cctgttggac gatgtcgaga aggccaccgg cgatcaatcg acaatggctg cacatttgaa 420caagtctcag gatgttggaa ttcatgcaag acatatcagc agtgtgtcta cattggatag 480tgaaatggct gaagacaatg ggaattcgat ttggaagaac agggtggaaa gttggaaaga 540aaagaagaac aagaagaaga agcctgcaac aactaaggtt gaaagagagg ctgaaatccc 600acctgagcaa caaatggaag ataaaccggc accggatgct tcccagcccc tctcgactat 660aattccaatc ccgaaaagca gacttgcacc ataccgaacc gtgatcatta tgcgattgat 720cattcttggt cttttcttcc attatcgagt aacaaacccc gttgacagtg cttttggact 780gtggctcact tcagtcatat gtgaaatctg gtttgcattt tcctgggtgt tggatcagtt 840ccctaagtgg tatcctgtta acagggaaac atacattgac agactatctg caagatatga 900aagagaaggt gaacctgatg aacttgctgc agttgacttc ttcgtgagta cagtggatcc 960attgaaagag cctccattga ttactgccaa tactgtgctt tccatccttg ccttggacta 1020cccggtggat aaggtctctt gttatatatc tgatgatggt gcggccatgc tgacatttga 1080atctctagta gaaacagccg actttgcaag aaagtgggtt ccattctgca aaaaattttc 1140cattgaaccc cgggcacctg agttttactt ctcacagaag attgattact tgaaagataa 1200agtgcagccc tcttttgtaa aagaacgtag agctatgaaa agagattatg aagagtacaa 1260aattcgaatc aatgctttag ttgcaaaggc tcagaaaaca cctgatgaag gatggacaat 1320gcaagatgga acttcttggc caggaaataa cccgcgtgat caccctggca tgattcaggt 1380tttccttgga tatagtggtg ctcgtgacat cgaaggaaat gaacttcctc gactggttta 1440cgtctctaga gagaagagac ctggctacca acaccacaaa aaggctggtg ctgaaaatgc 1500tttggttagg gtgtctgcag ttcttacaaa tgctcccttc atcctcaatc ttgattgtga 1560ccactatgtt aacaatagca aggcagttag ggaggcaatg tgcttcttga tggacccaca 1620agttggtcga gatgtatgct atgtgcagtt tcctcaaaga tttgatggca tagataggag 1680tgatcgatat gccaatagga acacagtttt ctttgatgtt aacatgaaag gtcttgatgg 1740aatccaaggg ccagtttatg tgggaacagg ttgtgttttc aataggcaag cactttatgg 1800ctatggtcca ccttcaatgc caagttttcc caagtcatcc tcctcatctt gctcgtgttg 1860ctgcccgggc aagaaggaac ctaaagatcc atcagagctt tatagggatg caaaacggga 1920agaacttgat gctgccatct ttaaccttag ggaaattgac aattatgatg agtatgaaag 1980atcaatgttg atctctcaaa caagctttga gaaaactttt ggcttatctt cagtcttcat 2040tgaatctaca ctaatggaga atggaggagt ggctgaatct gccaaccctt ccacactaat 2100caaggaagca attcatgtca tcagctgtgg ctatgaagag aagactgcat gggggaaaga 2160gattggatgg atatatggtt cagtcactga ggatatctta accggcttca aaatgcactg 2220ccgaggatgg agatcgattt actgcatgcc cttaaggcca gcattcaaag gatctgcacc 2280catcaatctg tctgatcggt tgcaccaggt tcttcgatgg gctcttggat ctgttgaaat 2340tttcctaagc aggcattgcc ctctatggta tggctttgga ggtggtcgtc ttaaatggct 2400tcaaagacta gcatatataa acaccattgt ctatcctttc acatcccttc cactcattgc 2460ctattgttca ctaccagcaa tctgtcttct cacaggaaaa tttatcatac caacgctctc 2520aaacctggca agtgttctct ttcttggcct tttcctttcc attatcgtga ctgctgttct 2580cgagctccga tggagtggtg tcagcattga ggacttatgg cgtaacgagc agttttgggt 2640catcggtggc gtttcagccc atctctttgc cgtcttccaa ggtttcctta agatgcttgc 2700gggcattgac accaacttta ctgtcactgc caaagcagct gatgatgcag attttggtga 2760gctctacatt gtgaaatgga ctacacttct aatccctcca acaacactcc tcatcgtcaa 2820catggttggt gtcgttgccg gattctccga tgccctcaac aaagggtacg aagcttgggg 2880accactcttt ggcaaagtgt tcttttcctt ctgggtcatc ctccatcttt atccattcct 2940caaaggtctt atgggacgcc aaaacaggac accaaccatt gttgtccttt ggtcagtgtt 3000gttggcttct gtcttctctc ttgtttgggt tcggatcaac ccgtttgtca gcaccgccga 3060tagcaccacc gtgtcacaga gctgcatttc cattgattgt tgatgatatt atgtgtttct 3120tagaattgaa atcattgcaa gtaagtggac tgaaacatgt ctattgacta agttttgaac 3180agtttgtacc cattttattc ttagcagtgt gtaattttcc taaacaatgc tatgaactat 3240acatatttca ttgatattta cattaaatga aactacatca gtctgcagaa aaaaaaaaaa 3300aaaaaaaaac tcgagggggg gcccggta 3328 2 4612 DNA Artificial SequenceSynthetic Oligonucleotide 2 aactagtgga tcccccgggc tgcaggaatt cggcacgagcgaggagatgg gttccgtttt 60 gtaagaagca ttgatcacct agggggcccg acgtccttaagccgtgctcg ctcctctacc 120 caaggcaaaa cattcttcgt taatgttgag cccagggcgccggagtttta tttcaatgag 180 aagattgatt atttgaagga caaggtccat attacaactcgggtcccgcg gcctcaaaat 240 aaagttactc ttctaactaa taaacttcct gttccaggtacctagctttg ttaaagaacg 300 gagagccatg aaaagggaat atgaagaatt taaagtaaggatcaatgcat ggatcgaaac 360 aatttcttgc ctctcggtac ttttccctta tacttcttaaatttcattcc tagttacgta 420 tagtagcaaa agctcagaag aaaccagaag aaggatgggtgatgcaagat ggcaccccat 480 ggcccggaaa atcatcgttt tcgagtcttc tttggtcttcttcctaccca ctacgttcta 540 ccgtggggta ccgggccttt taacactcgt gatcatcctggaatgattca ggtctatcta 600 ggaagtgccg gtgcactcga tgtggatggc attgtgagcactagtaggac cttactaagt 660 ccagatagat ccttcacggc cacgtgagct acacctaccgaaagagctgc ctcgacttgt 720 ctatgtttct cgtgagaaac gacctggtta tcagcaccataagaaagccg tttctcgacg 780 gagctgaaca gatacaaaga gcactctttg ctggaccaatagtcgtggta ttctttcggc 840 gtgctgagaa tgctctggtt cgagtttctg cagtgcttactaatgcaccc ttcatattga 900 atctggattg cacgactctt acgagaccaa gctcaaagacgtcacgaatg attacgtggg 960 aagtataact tagacctaac tgatcattac atcaacaatagcaaggccat gagggaagcg 1020 atgtgctttt taatggatcc tcagtttgga actagtaatgtagttgttat cgttccggta 1080 ctcccttcgc tacacgaaaa attacctagg agtcaaacctaagaagcttt gttatgttca 1140 atttccacag agatttgatg gtattgatcg tcatgatcgatatgctaatc ttcttcgaaa 1200 caatacaagt taaaggtgtc tctaaactac cataactagcagtactagct atacgattag 1260 gaaatgttgt cttctttgat atcaacatgt tgggattagatggacttcaa ggccctgtat 1320 atgtaggcac ctttacaaca gaagaaacta tagttgtacaaccctaatct acctgaagtt 1380 ccgggacata tacatccgtg agggtgtgtt ttcaacaggcaggcattgta tggctacgat 1440 ccaccagtct ctgagaaacg accaaagatg tcccacacaaaagttgtccg tccgtaacat 1500 accgatgcta ggtggtcaga gactctttgc tggtttctacacatgtgatt gctggccttc 1560 ttggtgttgc tgttgttgcg gaggttctag gaagaaatcaaagaagaaag tgtacactaa 1620 cgaccggaag aaccacaacg acaacaacgc ctccaagatccttctttagt ttcttctttc 1680 gtgaaaagaa gggcttactc ggaggtcttt tatacggaaaaaagaagaag atgatgggca 1740 aaaactatgt cacttttctt cccgaatgag cctccagaaaatatgccttt tttcttcttc 1800 tactacccgt ttttgataca gaaaaaaggg tctgcaccagtctttgatct cgaagaaatc 1860 gaagaagggc ttgaaggata cgaagaattg cttttttcccagacgtggtc agaaactaga 1920 gcttctttag cttcttcccg aacttcctat gcttcttaacgagaaatcga cattaatgtc 1980 gcagaagaat ttcgagaaac gattcggaca atcaccggttttcattgcct ctctttagct 2040 gtaattacag cgtcttctta aagctctttg ctaagcctgttagtggccaa aagtaacgga 2100 caactttgat ggaaaatggt ggccttcctg aaggaactaattccacatca ctgattaaag 2160 aggccattca gttgaaacta ccttttacca ccggaaggacttccttgatt aaggtgtagt 2220 gactaatttc tccggtaagt cgtaattagc tgtggttatgaagaaaaaac tgagtggggc 2280 aaagagatcg gatggattta tgggtcggtg gcattaatcgacaccaatac ttcttttttg 2340 actcaccccg tttctctagc ctacctaaat acccagccacacggaagata tattaacagg 2400 tttcaagatg cattgtagag ggtggaaatc ggtttattgtgtaccgaaaa tgccttctat 2460 ataattgtcc aaagttctac gtaacatctc ccacctttagccaaataaca catggctttt 2520 gaccggcatt caaagggtcc gctccaatca atctctcggatcggttgcac caagttttga 2580 gatgggcact ctggccgtaa gtttcccagg cgaggttagttagagagcct agccaacgtg 2640 gttcaaaact ctacccgtga tggttctgta gaaattttccttagtcgtca ctgtccactt 2700 tggtatggtt atggtggaaa actgaaatgg accaagacatctttaaaagg aatcagcagt 2760 gacaggtgaa accataccaa taccaccttt tgactttaccctcgagaggc ttgcttatat 2820 caacaccatt gtttaccctt tcacctcgat ccctttactcgcctattgta gagctctccg 2880 aacgaatata gttgtggtaa caaatgggaa agtggagctagggaaatgag cggataacat 2940 ctattccagc tgtttgtctt ctcaccggca aattcatcattccaactcta agcaacctta 3000 caagtgtgtg gataaggtcg acaaacagaa gagtggccgtttaagtagta aggttgagat 3060 tcgttggaat gttcacacac gttcttggca cttttcctctccatcattgc aactggagtg 3120 cttgaacttc gatggagcgg ggttagcatc caagaaccgtgaaaaggaga ggtagtaacg 3180 ttgacctcac gaacttgaag ctacctcgcc ccaatcgtagcaagactggt ggcgcaatga 3240 acaattctgg gtgatcggag gtgtctccgc ccatctttttgctgtcttcc gttctgacca 3300 ccgcgttact tgttaagacc cactagcctc cacagaggcgggtagaaaaa cgacagaagg 3360 agggcctcct caaagtccta gctggagtag acaccaacttcaccgtaaca gcaaaagcag 3420 cagacgatac tcccggagga gtttcaggat cgacctcatctgtggttgaa gtggcattgt 3480 cgttttcgtc gtctgctatg agaattcggt gaactttatctcttcaaatg gacaactctc 3540 ttaatccctc ccacaactct gataatactg tcttaagccacttgaaatag agaagtttac 3600 ctgttgagag aattagggag ggtgttgaga ctattatgacaacatggtcg gagtcgtggc 3660 cggagtttca gacgcaatca acaacggcta tggttcatggggtccattgt ttgtaccagc 3720 ctcagcaccg gcctcaaagt ctgcgttagt tgttgccgataccaagtacc ccaggtaaca 3780 tcggcaaact gttcttcgca ttctgggtca ttcttcatctttacccattc ctcaaaggtt 3840 tgatggggag agccgtttga caagaagcgt aagacccagtaagaagtaga aatgggtaag 3900 gagtttccaa actacccctc acaaaacagg acgcccaccattgttgtgct ttggtccata 3960 cttttggcat cgattttctc actggtttgg tgttttgtcctgcgggtggt aacaacacga 4020 aaccaggtat gaaaaccgta gctaaaagag tgaccaaaccgtacggatcg atcccttctt 4080 gcccaaacaa acaggtccag ttcttaaaca atgtggcgtggagtgctaaa catgcctagc 4140 tagggaagaa cgggtttgtt tgtccaggtc aagaatttgttacaccgcac ctcacgattt 4200 tggtgtttta caaacctttc ttattatttt attttccctttttgccacta ctgttgattt 4260 gctgtgattc accacaaaat gtttggaaag aataataaaataaaagggaa aaacggtgat 4320 gacaactaaa cgacactaag taaaagggat ttatcttgtttgtaaaaagt ctcctatgat 4380 tttgttggtt caatttaatt tctatatggt attttccctaaatagaacaa acatttttca 4440 gaggatacta aaacaaccaa gttaaattaa agatataccaaaaaaaatat ttctttaaat 4500 taactataaa aaaaaaaaaa aaaaactcga gggggggcccggtacctttt tttataaaga 4560 aatttaattg atattttttt tttttttttt tgagctcccccccgggccat gg 4612 3 1063 DNA Artificial Sequence SyntheticOligonucleotide 3 gggtgattga ctaaaatttt taaaaatttt gaaggtttta atgagaatttttaaacaatt 60 ttgtatgtta aactaaaact ttcaaaaaaa attttgaaag gtttaatgagaattttaaaa 120 attttgagcg ggctaattaa aatttttaaa aaatgtataa taaaaaaattcaaaaactct 180 ttgaggccat aaaggtcatc gggcccttaa atacatcagc ttgttgtttcctcatattac 240 tcatgttatt tcagttaaca gatataatgg ctatcatttg atttaggagtgaaatctaaa 300 aattcgaaaa gtataaaaac taaaaaggat taaattgaag aacattaattaaatcaacaa 360 tttactattc caataacaga attttgagtt aacaaattta actgctacaatttggttcga 420 gaccaaaatt acaaaacccg aaaagtattg ggactaaaat tgatcaaattagagtacatg 480 ggttaaattc acaacttact tatggtacaa ggattaatag cataatttctccttaggcaa 540 atgccagtta gttaaagatg taccttgccc aaccgaaagc ttccttaaacttcccgcaat 600 tttttaaatt tctttttccc ttagaaaaaa gaacaaaaat gtaagctttgcttgtcagag 660 atttctctgc aaatacattg acaccaacaa cctaccctcc attacactaccaaccggcct 720 tccccttcaa cttttcttca ccattacaac atgcctatct ccacccttagcccaacatgc 780 acttatatct tgtgtttggt tgtttttctt tttcatataa aaacacacaccaagacacaa 840 aggtattgag aggtaagtag agggaaagac cctttggtta gcatattgtttgtagcattg 900 ggttttttct caaggaagaa gaaggagaaa gataagtact ttttttgagaatgatggaat 960 ctggggttcc tgtttgccac acttgtggtg aacatgttgg gttgaatgtaagccgaattc 1020 cagcacactg gcggccgtta ctagtggatc cgcgctcggt acc 1063 427 DNA Artificial Sequence Synthetic Oligonucleotide 4 attgaattcctgggtgttgg atcagtt 27 5 24 DNA Artificial Sequence SyntheticOligonucleotide 5 attctcgagt ggaagggatt gaaa 24 6 974 PRT Gossypimhirsutum 6 Met Met Glu Ser Gly Val Pro Val Cys His Thr Cys Gly Glu HisVal 1 5 10 15 Gly Leu Asn Val Asn Gly Glu Pro Phe Val Ala Cys His GluCys Asn 20 25 30 Phe Pro Ile Cys Lys Ser Cys Phe Glu Tyr Asp Leu Lys GluGly Arg 35 40 45 Lys Ala Cys Leu Arg Cys Gly Ser Pro Tyr Asp Glu Asn LeuLeu Asp 50 55 60 Asp Val Glu Lys Ala Thr Gly Asp Gln Ser Thr Met Ala AlaHis Leu 65 70 75 80 Asn Lys Ser Gln Asp Val Gly Ile His Ala Arg His IleSer Ser Val 85 90 95 Ser Thr Leu Asp Ser Glu Met Ala Glu Asp Asn Gly AsnSer Ile Trp 100 105 110 Lys Asn Arg Val Glu Ser Trp Lys Glu Lys Lys AsnLys Lys Lys Lys 115 120 125 Pro Ala Thr Thr Lys Val Glu Arg Glu Ala GluIle Pro Pro Glu Gln 130 135 140 Gln Met Glu Asp Lys Pro Ala Pro Asp AlaSer Gln Pro Leu Ser Thr 145 150 155 160 Ile Ile Pro Ile Pro Lys Ser ArgLeu Ala Pro Tyr Arg Thr Val Ile 165 170 175 Ile Met Arg Leu Ile Ile LeuGly Leu Phe Phe His Tyr Arg Val Thr 180 185 190 Asn Pro Val Asp Ser AlaPhe Gly Leu Trp Leu Thr Ser Val Ile Cys 195 200 205 Glu Ile Trp Phe AlaPhe Ser Trp Val Leu Asp Gln Phe Pro Lys Trp 210 215 220 Tyr Pro Val AsnArg Glu Thr Tyr Ile Asp Arg Leu Ser Ala Arg Tyr 225 230 235 240 Glu ArgGlu Gly Glu Pro Asp Glu Leu Ala Ala Val Asp Phe Phe Val 245 250 255 SerThr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn Thr 260 265 270Val Leu Ser Ile Leu Ala Leu Asp Tyr Pro Val Asp Lys Val Ser Cys 275 280285 Tyr Ile Ser Asp Asp Gly Ala Ala Met Leu Thr Phe Glu Ser Leu Val 290295 300 Glu Thr Ala Asp Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Phe305 310 315 320 Ser Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln LysIle Asp 325 330 335 Tyr Leu Lys Asp Lys Val Gln Pro Ser Phe Val Lys GluArg Arg Ala 340 345 350 Met Lys Arg Asp Tyr Glu Glu Tyr Lys Ile Arg IleAsn Ala Leu Val 355 360 365 Ala Lys Ala Gln Lys Thr Pro Asp Glu Gly TrpThr Met Gln Asp Gly 370 375 380 Thr Ser Trp Pro Gly Asn Asn Pro Arg AspHis Pro Gly Met Ile Gln 385 390 395 400 Val Phe Leu Gly Tyr Ser Gly AlaArg Asp Ile Glu Gly Asn Glu Leu 405 410 415 Pro Arg Leu Val Tyr Val SerArg Glu Lys Arg Pro Gly Tyr Gln His 420 425 430 His Lys Lys Ala Gly AlaGlu Asn Ala Leu Val Arg Val Ser Ala Val 435 440 445 Leu Thr Asn Ala ProPhe Ile Leu Asn Leu Asp Cys Asp His Tyr Val 450 455 460 Asn Asn Ser LysAla Val Arg Glu Ala Met Cys Phe Leu Met Asp Pro 465 470 475 480 Gln ValGly Arg Asp Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp 485 490 495 GlyIle Asp Arg Ser Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe 500 505 510Asp Val Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val 515 520525 Gly Thr Gly Cys Val Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Gly Pro 530535 540 Pro Ser Met Pro Ser Phe Pro Lys Ser Ser Ser Ser Ser Cys Ser Cys545 550 555 560 Cys Cys Pro Gly Lys Lys Glu Pro Lys Asp Pro Ser Glu LeuTyr Arg 565 570 575 Asp Ala Lys Arg Glu Glu Leu Asp Ala Ala Ile Phe AsnLeu Arg Glu 580 585 590 Ile Asp Asn Tyr Asp Glu Tyr Glu Arg Ser Met LeuIle Ser Gln Thr 595 600 605 Ser Phe Glu Lys Thr Phe Gly Leu Ser Ser ValPhe Ile Glu Ser Thr 610 615 620 Leu Met Glu Asn Gly Gly Val Ala Glu SerAla Asn Pro Ser Thr Leu 625 630 635 640 Ile Lys Glu Ala Ile His Val IleSer Cys Gly Tyr Glu Glu Lys Thr 645 650 655 Ala Trp Gly Lys Glu Ile GlyTrp Ile Tyr Gly Ser Val Thr Glu Asp 660 665 670 Ile Leu Thr Gly Phe LysMet His Cys Arg Gly Trp Arg Ser Ile Tyr 675 680 685 Cys Met Pro Leu ArgPro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu 690 695 700 Ser Asp Arg LeuHis Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu 705 710 715 720 Ile PheLeu Ser Arg His Cys Pro Leu Trp Tyr Gly Phe Gly Gly Gly 725 730 735 ArgLeu Lys Trp Leu Gln Arg Leu Ala Tyr Ile Asn Thr Ile Val Tyr 740 745 750Pro Phe Thr Ser Leu Pro Leu Ile Ala Tyr Cys Ser Leu Pro Ala Ile 755 760765 Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr Leu Ser Asn Leu Ala 770775 780 Ser Val Leu Phe Leu Gly Leu Phe Leu Ser Ile Ile Val Thr Ala Val785 790 795 800 Leu Glu Leu Arg Trp Ser Gly Val Ser Ile Glu Asp Leu TrpArg Asn 805 810 815 Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His LeuPhe Ala Val 820 825 830 Phe Gln Gly Phe Leu Lys Met Leu Ala Gly Ile AspThr Asn Phe Thr 835 840 845 Val Thr Ala Lys Ala Ala Asp Asp Ala Asp PheGly Glu Leu Tyr Ile 850 855 860 Val Lys Trp Thr Thr Leu Leu Ile Pro ProThr Thr Leu Leu Ile Val 865 870 875 880 Asn Met Val Gly Val Val Ala GlyPhe Ser Asp Ala Leu Asn Lys Gly 885 890 895 Tyr Glu Ala Trp Gly Pro LeuPhe Gly Lys Val Phe Phe Ser Phe Trp 900 905 910 Val Ile Leu His Leu TyrPro Phe Leu Lys Gly Leu Met Gly Arg Gln 915 920 925 Asn Arg Thr Pro ThrIle Val Val Leu Trp Ser Val Leu Leu Ala Ser 930 935 940 Val Phe Ser LeuVal Trp Val Arg Ile Asn Pro Phe Val Ser Thr Ala 945 950 955 960 Asp SerThr Thr Val Ser Gln Ser Cys Ile Ser Ile Asp Cys 965 970 7 685 PRTGossypium hirsutum 7 Ala Arg Arg Trp Val Pro Phe Cys Lys Lys His Asn ValGlu Pro Arg 1 5 10 15 Ala Pro Glu Phe Tyr Phe Asn Glu Lys Ile Asp TyrLeu Lys Asp Lys 20 25 30 Val His Pro Ser Phe Val Lys Glu Arg Arg Ala MetLys Arg Glu Tyr 35 40 45 Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val AlaLys Ala Gln Lys 50 55 60 Lys Pro Glu Glu Gly Trp Val Met Gln Asp Gly ThrPro Trp Pro Gly 65 70 75 80 Asn Asn Thr Arg Asp His Pro Gly Met Ile GlnVal Tyr Leu Gly Ser 85 90 95 Ala Gly Ala Leu Asp Val Asp Gly Lys Glu LeuPro Arg Leu Val Tyr 100 105 110 Val Ser Arg Glu Lys Arg Pro Gly Tyr GlnHis His Lys Lys Ala Gly 115 120 125 Ala Glu Asn Ala Leu Val Arg Val SerAla Val Leu Thr Asn Ala Pro 130 135 140 Phe Ile Leu Asn Leu Asp Cys AspHis Tyr Ile Asn Asn Ser Lys Ala 145 150 155 160 Met Arg Glu Ala Met CysPhe Leu Met Asp Pro Gln Phe Gly Lys Lys 165 170 175 Leu Cys Tyr Val GlnPhe Pro Gln Arg Phe Asp Gly Ile Asp Arg His 180 185 190 Asp Arg Tyr AlaAsn Arg Asn Val Val Phe Phe Asp Ile Asn Met Leu 195 200 205 Gly Leu AspGly Leu Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Val 210 215 220 Phe AsnArg Gln Ala Leu Tyr Gly Tyr Asp Pro Pro Val Ser Glu Lys 225 230 235 240Arg Pro Lys Met Thr Cys Asp Cys Trp Pro Ser Trp Cys Cys Cys Cys 245 250255 Cys Gly Gly Ser Arg Lys Lys Ser Lys Lys Lys Gly Glu Lys Lys Gly 260265 270 Leu Leu Gly Gly Leu Leu Tyr Gly Lys Lys Lys Lys Met Met Gly Lys275 280 285 Asn Tyr Val Lys Lys Gly Ser Ala Pro Val Phe Asp Leu Glu GluIle 290 295 300 Glu Glu Gly Leu Glu Gly Tyr Glu Glu Leu Glu Lys Ser ThrLeu Met 305 310 315 320 Ser Gln Lys Asn Phe Glu Lys Arg Phe Gly Gln SerPro Val Phe Ile 325 330 335 Ala Ser Thr Leu Met Glu Asn Gly Gly Leu ProGlu Gly Thr Asn Ser 340 345 350 Thr Ser Leu Ile Lys Glu Ala Ile His ValIle Ser Cys Gly Tyr Glu 355 360 365 Glu Lys Thr Glu Trp Gly Lys Glu IleGly Trp Ile Tyr Gly Ser Val 370 375 380 Thr Glu Asp Ile Leu Thr Gly PheLys Met His Cys Arg Gly Trp Lys 385 390 395 400 Ser Val Tyr Cys Val ProLys Arg Pro Ala Phe Lys Gly Ser Ala Pro 405 410 415 Ile Asn Leu Ser AspArg Leu His Gln Val Leu Arg Trp Ala Leu Gly 420 425 430 Ser Val Glu IlePhe Leu Ser Arg His Cys Pro Leu Trp Tyr Gly Tyr 435 440 445 Gly Gly LysLeu Lys Trp Leu Glu Arg Leu Ala Tyr Ile Asn Thr Ile 450 455 460 Val TyrPro Phe Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Ile Pro 465 470 475 480Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr Leu Ser Asn 485 490495 Leu Thr Ser Val Trp Phe Leu Ala Leu Phe Leu Ser Ile Ile Ala Thr 500505 510 Gly Val Leu Glu Leu Arg Trp Ser Gly Val Ser Ile Gln Asp Trp Trp515 520 525 Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His LeuPhe 530 535 540 Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Val AspThr Asn 545 550 555 560 Phe Thr Val Thr Ala Lys Ala Ala Asp Asp Thr GluPhe Gly Glu Leu 565 570 575 Tyr Leu Phe Lys Trp Thr Thr Leu Leu Ile ProPro Thr Thr Leu Ile 580 585 590 Ile Leu Asn Met Val Gly Val Val Ala GlyVal Ser Asp Ala Ile Asn 595 600 605 Asn Gly Tyr Gly Ser Trp Gly Pro LeuPhe Gly Lys Leu Phe Phe Ala 610 615 620 Phe Trp Val Ile Leu His Leu TyrPro Phe Leu Lys Gly Leu Met Gly 625 630 635 640 Arg Gln Asn Arg Thr ProThr Ile Val Val Leu Trp Ser Ile Leu Leu 645 650 655 Ala Ser Ile Phe SerLeu Val Trp Val Arg Ile Asp Pro Phe Leu Pro 660 665 670 Lys Gln Thr GlyPro Val Leu Lys Gln Cys Gly Val Glu 675 680 685 8 881 PRT Oryzae sativa8 Gly Asn Val Ala Trp Lys Glu Arg Val Asp Gly Trp Lys Leu Lys Gln 1 5 1015 Asp Lys Gly Ala Ile Pro Met Thr Asn Gly Thr Ser Ile Ala Pro Ser 20 2530 Glu Gly Arg Gly Val Gly Asp Ile Asp Ala Ser Thr Asp Tyr Asn Asn 35 4045 Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg Gln Pro Leu Ser Arg Lys 50 5560 Val Pro Leu Pro Ser Ser Arg Ile Asn Pro Tyr Arg Asn Val Ile Val 65 7075 80 Leu Arg Leu Val Val Leu Ser Ile Phe Leu His Tyr Arg Ile Thr Asn 8590 95 Pro Val Arg Asn Ala Tyr Pro Leu Trp Leu Leu Ser Val Ile Cys Glu100 105 110 Ile Trp Phe Ala Leu Ser Trp Leu Ile Asp Gln Phe Pro Lys TrpPhe 115 120 125 Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu ArgTyr Asp 130 135 140 Arg Glu Gly Glu Pro Ser Gln Leu Ala Ala Val Asp IlePhe Val Ser 145 150 155 160 Thr Val Asp Pro Met Lys Glu Pro Pro Leu ValThr Ala Asn Thr Val 165 170 175 Leu Ser Ile Leu Ala Val Asp Tyr Pro ValAsp Lys Val Ser Cys Tyr 180 185 190 Val Ser Asp Asp Gly Ala Ala Met LeuThr Phe Asp Ala Leu Ala Glu 195 200 205 Thr Ser Glu Phe Ala Arg Lys TrpVal Pro Phe Val Lys Lys Tyr Asn 210 215 220 Ile Glu Pro Arg Ala Pro GluTrp Tyr Phe Ser Gln Lys Ile Asp Tyr 225 230 235 240 Leu Lys Asp Lys ValHis Pro Ser Phe Val Lys Asp Arg Arg Ala Met 245 250 255 Lys Arg Glu TyrGlu Glu Phe Lys Val Arg Ile Asn Gly Leu Val Ala 260 265 270 Lys Ala GlnLys Val Pro Glu Glu Gly Trp Ile Met Gln Asp Gly Thr 275 280 285 Pro TrpPro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val 290 295 300 PheLeu Gly His Ser Gly Gly Leu Asp Thr Glu Gly Asn Glu Leu Pro 305 310 315320 Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His 325330 335 Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg Val Ser Ala Val Leu340 345 350 Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp Cys Asp His Tyr IleAsn 355 360 365 Asn Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Leu Met AspPro Asn 370 375 380 Leu Gly Arg Ser Val Cys Tyr Val Gln Phe Pro Gln ArgPhe Asp Gly 385 390 395 400 Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg AsnThr Val Phe Phe Asp 405 410 415 Ile Asn Leu Arg Gly Leu Asp Gly Ile GlnGly Pro Val Tyr Val Gly 420 425 430 Thr Gly Cys Val Phe Asn Arg Thr AlaLeu Tyr Gly Tyr Glu Pro Pro 435 440 445 Ile Lys Gln Lys Lys Lys Gly SerPhe Leu Ser Ser Leu Cys Gly Gly 450 455 460 Arg Lys Lys Ala Ser Lys SerLys Lys Lys Ser Ser Asp Lys Lys Lys 465 470 475 480 Ser Asn Lys His ValAsp Ser Ala Val Pro Val Phe Asn Leu Glu Asp 485 490 495 Ile Glu Glu GlyVal Glu Gly Ala Gly Phe Asp Asp Glu Lys Ser Leu 500 505 510 Leu Met SerGln Met Ser Leu Glu Lys Arg Phe Gly Gln Ser Ala Ala 515 520 525 Phe ValAla Ser Thr Leu Met Glu Tyr Gly Gly Val Pro Gln Ser Ala 530 535 540 ThrPro Glu Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 545 550 555560 Tyr Glu Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly Trp Ile Tyr Gly 565570 575 Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly580 585 590 Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala Phe Lys GlySer 595 600 605 Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu ArgTrp Ala 610 615 620 Leu Gly Ser Val Glu Ile Leu Phe Ser Arg His Cys ProIle Trp Tyr 625 630 635 640 Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu ArgPhe Ala Tyr Ile Asn 645 650 655 Thr Thr Ile Tyr Pro Leu Thr Ser Ile ProLeu Leu Ile Tyr Cys Val 660 665 670 Leu Pro Ala Ile Cys Leu Leu Thr GlyLys Phe Ile Ile Pro Glu Ile 675 680 685 Ser Asn Phe Ala Ser Ile Trp PheIle Ser Leu Phe Ile Ser Ile Phe 690 695 700 Ala Thr Gly Ile Leu Glu MetArg Trp Ser Gly Val Gly Ile Asp Glu 705 710 715 720 Trp Trp Arg Asn GluGln Phe Trp Val Ile Gly Gly Ile Ser Ala His 725 730 735 Leu Phe Ala ValPhe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 740 745 750 Thr Asn PheThr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe 755 760 765 Ala GluLeu Tyr Met Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr 770 775 780 ThrIle Leu Ile Ile Asn Leu Val Gly Val Val Ala Gly Ile Ser Tyr 785 790 795800 Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 805810 815 Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly820 825 830 Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Val Val TrpAla 835 840 845 Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg IleAsp Pro 850 855 860 Phe Thr Thr Arg Val Thr Gly Pro Asp Thr Gln Thr CysGly Ile Asn 865 870 875 880 Cys 9 723 PRT Acetobacter xylinum 9 Met ProGlu Val Arg Ser Ser Thr Gln Ser Glu Ser Gly Met Ser Gln 1 5 10 15 TrpMet Gly Lys Ile Leu Ser Ile Arg Gly Ala Gly Leu Thr Ile Gly 20 25 30 ValPhe Gly Leu Cys Ala Leu Ile Ala Ala Thr Ser Val Thr Leu Pro 35 40 45 ProGlu Gln Gln Leu Ile Val Ala Phe Val Cys Val Val Ile Phe Phe 50 55 60 IleVal Gly His Lys Pro Ser Arg Arg Ser Gln Ile Phe Leu Glu Val 65 70 75 80Leu Ser Gly Leu Val Ser Leu Arg Tyr Leu Thr Trp Arg Leu Thr Glu 85 90 95Thr Leu Ser Phe Asp Thr Trp Leu Gln Gly Leu Leu Gly Thr Met Leu 100 105110 Leu Val Ala Glu Leu Tyr Ala Leu Met Met Leu Phe Leu Ser Tyr Phe 115120 125 Gln Thr Ile Ala Pro Leu His Arg Ala Pro Leu Pro Leu Pro Pro Asn130 135 140 Pro Asp Glu Trp Pro Thr Val Asp Ile Phe Val Pro Thr Tyr AsnGlu 145 150 155 160 Glu Leu Ser Ile Val Arg Leu Thr Val Leu Gly Ser LeuGly Ile Asp 165 170 175 Trp Pro Pro Glu Lys Val Arg Val His Ile Leu AspAsp Gly Arg Arg 180 185 190 Pro Glu Phe Ala Ala Phe Ala Ala Glu Cys GlyAla Asn Tyr Ile Ala 195 200 205 Arg Pro Thr Asn Glu His Ala Lys Ala GlyAsn Leu Asn Tyr Ala Ile 210 215 220 Gly His Thr Asp Gly Asp Tyr Ile LeuIle Phe Asp Cys Asp His Val 225 230 235 240 Pro Thr Arg Ala Phe Leu GlnLeu Thr Met Gly Trp Met Val Glu Asp 245 250 255 Pro Lys Ile Ala Leu MetGln Thr Pro His His Phe Tyr Ser Pro Asp 260 265 270 Pro Phe Gln Arg AsnLeu Ser Ala Gly Tyr Arg Thr Pro Pro Glu Gly 275 280 285 Asn Leu Phe TyrGly Val Val Gln Asp Gly Asn Asp Phe Trp Asp Ala 290 295 300 Thr Phe PheCys Gly Ser Cys Ala Ile Leu Arg Arg Thr Ala Ile Glu 305 310 315 320 GlnIle Gly Gly Phe Ala Thr Gln Thr Val Thr Glu Asp Ala His Thr 325 330 335Ala Leu Lys Met Gln Arg Leu Gly Trp Ser Thr Ala Tyr Leu Arg Ile 340 345350 Pro Leu Ala Gly Gly Leu Ala Thr Glu Arg Leu Ile Leu His Ile Gly 355360 365 Gln Arg Val Arg Trp Ala Arg Gly Met Leu Gln Ile Phe Arg Ile Asp370 375 380 Asn Pro Leu Phe Gly Arg Gly Leu Ser Trp Gly Gln Arg Leu CysTyr 385 390 395 400 Leu Ser Ala Met Thr Ser Phe Leu Phe Ala Val Pro ArgVal Ile Phe 405 410 415 Leu Ser Ser Pro Leu Ala Phe Leu Phe Phe Gly GlnAsn Ile Ile Ala 420 425 430 Ala Ser Pro Leu Ala Leu Leu Ala Tyr Ala IlePro His Met Phe His 435 440 445 Ala Val Gly Thr Ala Ser Lys Ile Asn LysGly Trp Arg Tyr Ser Phe 450 455 460 Trp Ser Glu Val Tyr Glu Thr Thr MetAla Leu Phe Leu Val Arg Val 465 470 475 480 Thr Ile Val Thr Leu Leu SerPro Ser Arg Gly Lys Phe Asn Val Thr 485 490 495 Asp Lys Gly Gly Leu LeuGlu Lys Gly Tyr Phe Asp Leu Gly Ala Val 500 505 510 Tyr Pro Asn Ile IleLeu Gly Leu Ile Met Phe Gly Gly Leu Ala Arg 515 520 525 Gly Val Tyr GluLeu Ser Phe Gly His Leu Asp Gln Ile Ala Glu Arg 530 535 540 Ala Tyr LeuLeu Asn Ser Ala Trp Ala Met Leu Ser Leu Ile Ile Ile 545 550 555 560 LeuAla Ala Ile Ala Val Gly Arg Glu Thr Gln Gln Lys Arg Asn Ser 565 570 575His Arg Ile Pro Ala Thr Ile Pro Val Glu Val Ala Asn Ala Asp Gly 580 585590 Ser Ile Ile Val Thr Gly Val Thr Glu Asp Leu Ser Met Gly Gly Ala 595600 605 Ala Val Lys Met Ser Trp Pro Ala Lys Leu Ser Gly Pro Thr Pro Val610 615 620 Tyr Ile Arg Thr Val Leu Asp Gly Glu Glu Leu Ile Leu Pro AlaArg 625 630 635 640 Ile Ile Arg Ala Gly Asn Gly Arg Gly Ile Phe Ile TrpThr Ile Asp 645 650 655 Asn Leu Gln Gln Glu Phe Ser Val Ile Arg Leu ValPhe Gly Arg Ala 660 665 670 Asp Ala Trp Val Asp Leu Gly Gln Leu Gln GlyArg Pro Pro Ala Ala 675 680 685 Gln Pro His Gly His Gly Ser Gln Arg GlnGly Pro Val Pro Phe Lys 690 695 700 Trp Arg Tyr Arg Pro Ser Gln Phe ProAsn Gln Ala Phe Gly Trp Gln 705 710 715 720 Cys Pro Val 10 756 PRTacetobacter xylinum 10 Met Ser Glu Val Gln Ser Pro Val Pro Thr Glu SerArg Leu Gly Arg 1 5 10 15 Ile Ser Asn Lys Ile Leu Ser Leu Arg Gly AlaSer Tyr Ile Val Gly 20 25 30 Ala Leu Gly Leu Cys Ala Leu Ile Ala Ala ThrThr Val Thr Leu Asn 35 40 45 Asn Asn Glu Gln Leu Ile Val Ala Ala Val CysVal Val Ile Phe Phe 50 55 60 Val Val Gly Arg Gly Lys Ser Arg Arg Thr GlnIle Phe Leu Glu Val 65 70 75 80 Leu Ser Ala Leu Val Ser Leu Arg Tyr LeuThr Trp Arg Leu Thr Glu 85 90 95 Thr Leu Asp Phe Asn Thr Trp Ile Gln GlyIle Leu Gly Val Ile Leu 100 105 110 Leu Met Ala Glu Leu Tyr Ala Leu TyrMet Leu Phe Leu Ser Tyr Phe 115 120 125 Gln Thr Ile Gln Pro Leu His ArgAla Pro Leu Pro Leu Pro Asp Asn 130 135 140 Val Asp Asp Trp Pro Thr ValAsp Ile Phe Ile Pro Thr Tyr Asp Glu 145 150 155 160 Gln Leu Ser Ile ValArg Leu Thr Val Leu Gly Ala Leu Gly Ile Asp 165 170 175 Trp Pro Pro AspLys Val Asn Val Tyr Ile Leu Asp Asp Gly Val Arg 180 185 190 Pro Glu PheGlu Gln Phe Ala Lys Asp Cys Gly Ala Leu Tyr Ile Gly 195 200 205 Arg ValAsp Val Asp Ser Ala His Ala Lys Ala Gly Asn Leu Asn His 210 215 220 AlaIle Lys Arg Thr Ser Gly Asp Tyr Ile Leu Ile Leu Asp Cys Asp 225 230 235240 His Ile Pro Thr Arg Ala Phe Leu Gln Ile Ala Met Gly Trp Met Val 245250 255 Ala Asp Arg Lys Ile Ala Leu Met Gln Thr Pro His His Phe Tyr Ser260 265 270 Pro Asp Pro Phe Gln Arg Asn Leu Ala Val Gly Tyr Arg Thr ProPro 275 280 285 Glu Gly Asn Leu Phe Tyr Gly Val Ile Gln Asp Gly Asn AspPhe Trp 290 295 300 Asp Ala Thr Phe Phe Cys Gly Ser Cys Ala Ile Leu ArgArg Glu Ala 305 310 315 320 Ile Glu Ser Ile Gly Gly Phe Ala Val Glu ThrVal Thr Glu Asp Ala 325 330 335 His Thr Ala Leu Arg Met Gln Arg Arg GlyTrp Ser Thr Ala Tyr Leu 340 345 350 Arg Ile Pro Val Ala Ser Gly Leu AlaThr Glu Arg Leu Thr Thr His 355 360 365 Ile Gly Gln Arg Met Arg Trp AlaArg Gly Met Ile Gln Ile Phe Arg 370 375 380 Val Asp Asn Pro Met Leu GlyArg Gly Leu Lys Leu Gly Gln Arg Leu 385 390 395 400 Cys Tyr Leu Ser AlaMet Thr Ser Phe Phe Phe Ala Ile Pro Arg Val 405 410 415 Ile Phe Leu AlaSer Pro Leu Ala Phe Leu Phe Ala Gly Gln Asn Ile 420 425 430 Ile Ala AlaAla Pro Leu Ala Val Ala Ala Tyr Ala Leu Pro His Met 435 440 445 Phe HisSer Ile Ala Thr Ala Ala Lys Val Asn Lys Gly Trp Arg Tyr 450 455 460 SerPhe Trp Ser Glu Val Tyr Glu Thr Thr Met Ala Leu Phe Leu Val 465 470 475480 Arg Val Thr Ile Val Thr Leu Leu Phe Pro Ser Lys Gly Lys Phe Asn 485490 495 Val Thr Glu Lys Gly Gly Val Leu Glu Glu Glu Glu Phe Asp Leu Gly500 505 510 Ala Thr Tyr Pro Asn Ile Ile Phe Ala Thr Ile Met Met Gly GlyLeu 515 520 525 Leu Ile Gly Leu Phe Glu Leu Ile Val Arg Phe Asn Gln LeuAsp Val 530 535 540 Ile Ala Arg Asn Ala Tyr Leu Leu Asn Cys Ala Trp AlaLeu Ile Ser 545 550 555 560 Leu Ile Ile Leu Phe Ala Ala Ile Ala Val GlyArg Glu Thr Lys Gln 565 570 575 Val Arg Tyr Asn His Arg Val Glu Ala HisIle Pro Val Thr Val Tyr 580 585 590 Asp Ala Pro Ala Glu Gly Gln Pro HisThr Tyr Tyr Asn Ala Thr His 595 600 605 Gly Met Thr Gln Asp Val Ser MetGly Gly Val Ala Val His Ile Pro 610 615 620 Leu Pro Asp Val Thr Thr GlyPro Val Lys Lys Arg Ile His Ala Val 625 630 635 640 Leu Asp Gly Glu GluIle Asp Ile Pro Ala Thr Met Leu Arg Cys Thr 645 650 655 Asn Gly Lys AlaVal Phe Thr Trp Asp Asn Asn Asp Leu Asp Thr Glu 660 665 670 Arg Asp IleVal Arg Phe Val Phe Gly Arg Ala Asp Ala Trp Leu Gln 675 680 685 Trp AsnAsn Tyr Glu Asp Asp Arg Pro Leu Arg Ser Leu Trp Ser Leu 690 695 700 LeuLeu Ser Ile Lys Ala Leu Phe Arg Lys Lys Gly Lys Ile Met Ala 705 710 715720 Asn Ser Arg Pro Lys Lys Lys Pro Leu Ala Leu Pro Val Glu Arg Arg 725730 735 Glu Pro Thr Thr Ile His Ser Gly Gln Thr Gln Glu Gly Lys Ile Ser740 745 750 Arg Ala Ala Ser 755 11 693 PRT Escherichia coli 11 Met LeuLeu Trp Gly Val Ala Leu Ile Val Arg Arg Met Pro Gly Arg 1 5 10 15 PheSer Ala Leu Met Leu Ile Val Leu Ser Leu Thr Val Ser Cys Arg 20 25 30 TyrIle Trp Trp Arg Tyr Thr Ser Thr Leu Asn Trp Asp Asp Pro Val 35 40 45 SerLeu Val Cys Gly Leu Ile Leu Leu Phe Ala Ile Thr Tyr Ala Trp 50 55 60 IleVal Leu Val Leu Gly Tyr Phe Gln Val Val Trp Pro Leu Asn Arg 65 70 75 80Gln Pro Val Pro Leu Pro Lys Asp Met Ser Leu Trp Pro Ser Val Asp 85 90 95Ile Phe Val Pro Thr Tyr Asn Glu Asp Leu Asn Val Val Lys Asn Thr 100 105110 Ile Tyr Ala Ser Leu Gly Ile Asp Trp Pro Lys Asp Lys Leu Asn Ile 115120 125 Trp Ile Leu Asp Asp Gly Gly Arg Glu Glu Phe Arg Gln Phe Ala Gln130 135 140 Asn Val Gly Val Lys Tyr Ile Ala Arg Thr Thr His Glu His AlaLys 145 150 155 160 Ala Gly Asn Ile Asn Asn Ala Leu Lys Tyr Ala Lys GlyGlu Phe Val 165 170 175 Ser Ile Phe Asp Cys Asp His Val Pro Thr Arg SerPhe Leu Gln Met 180 185 190 Thr Met Gly Trp Phe Leu Lys Glu Lys Gln LeuAla Met Met Gln Thr 195 200 205 Pro His His Phe Phe Ser Pro Asp Pro PheGlu Arg Asn Leu Gly Arg 210 215 220 Phe Arg Lys Thr Pro Asn Glu Gly ThrLeu Phe Tyr Gly Leu Val Gln 225 230 235 240 Asp Gly Asn Asp Met Trp AspAla Thr Phe Phe Cys Gly Ser Cys Ala 245 250 255 Val Ile Arg Arg Lys ProLeu Asp Glu Ile Gly Gly Ile Ala Val Glu 260 265 270 Thr Val Thr Glu AspAla His Thr Ser Leu Arg Leu His Arg Arg Gly 275 280 285 Tyr Thr Ser AlaTyr Met Arg Ile Pro Gln Ala Ala Gly Leu Ala Thr 290 295 300 Glu Ser LeuSer Ala His Ile Gly Gln Arg Ile Arg Trp Ala Arg Gly 305 310 315 320 MetVal Gln Ile Phe Arg Leu Asp Asn Pro Leu Thr Gly Lys Gly Leu 325 330 335Lys Phe Ala Gln Arg Leu Cys Tyr Val Asn Ala Met Phe His Phe Leu 340 345350 Ser Gly Ile Pro Arg Leu Ile Phe Leu Thr Ala Pro Leu Ala Phe Leu 355360 365 Leu Leu His Ala Tyr Ile Ile Tyr Ala Pro Ala Leu Met Ile Ala Leu370 375 380 Phe Val Leu Pro His Met Ile His Ala Ser Leu Thr Asn Ser LysIle 385 390 395 400 Gln Gly Lys Tyr Arg His Ser Phe Trp Ser Glu Ile TyrGlu Thr Val 405 410 415 Leu Ala Trp Tyr Ile Ala Pro Pro Thr Leu Val AlaLeu Ile Asn Pro 420 425 430 His Lys Gly Lys Phe Asn Val Thr Ala Lys GlyGly Gly Leu Val Glu 435 440 445 Glu Glu Tyr Val Asp Trp Val Ile Ser ArgPro Tyr Ile Phe Leu Val 450 455 460 Leu Leu Asn Leu Val Gly Val Ala ValGly Ile Trp Arg Tyr Phe Tyr 465 470 475 480 Gly Pro Pro Thr Glu Met LeuThr Val Val Val Ser Met Val Trp Val 485 490 495 Phe Tyr Asn Leu Ile ValLeu Gly Gly Ala Val Ala Val Ser Val Glu 500 505 510 Ser Lys Gln Val ArgArg Ser His Arg Val Glu Met Thr Met Pro Ala 515 520 525 Ala Ile Ala ArgGlu Asp Gly His Leu Phe Ser Cys Thr Val Gln Asp 530 535 540 Phe Ser AspGly Gly Leu Gly Ile Lys Ile Asn Gly Gln Ala Gln Ile 545 550 555 560 LeuGlu Gly Gln Lys Val Asn Leu Leu Leu Lys Arg Gly Gln Gln Glu 565 570 575Tyr Val Phe Pro Thr Gln Val Ala Arg Val Met Gly Asn Glu Val Gly 580 585590 Leu Lys Leu Met Pro Leu Thr Thr Gln Gln His Ile Asp Phe Val Gln 595600 605 Cys Thr Phe Ala Arg Ala Asp Thr Trp Ala Leu Trp Gln Asp Ser Tyr610 615 620 Pro Glu Asp Lys Pro Leu Glu Ser Leu Leu Asp Ile Leu Lys LeuGly 625 630 635 640 Phe Arg Gly Tyr Arg His Leu Ala Glu Phe Ala Pro SerSer Val Lys 645 650 655 Gly Ile Phe Arg Val Leu Thr Ser Leu Val Ser TrpVal Val Ser Phe 660 665 670 Ile Pro Pro Arg Pro Glu Arg Ser Glu Thr AlaGln Pro Ser Asp Gln 675 680 685 Ala Leu Ala Gln Gln 690 12 861 PRTAgrobacterium tumefaciens 12 Met Cys Arg Cys Gly Arg Ala Val Arg Ser ArgPro Val Cys Arg Pro 1 5 10 15 Gly Gln Leu Val Val Arg Arg Ser Pro ArgPro Arg Ser Arg Asn His 20 25 30 Ser Arg Cys Arg Pro Leu Arg Leu Ser ValPhe Pro Arg Pro His Arg 35 40 45 Arg Val Arg His His Cys Gln Arg Asp LeuArg Trp Glu Pro Gly Arg 50 55 60 Trp Ile Ala Val Arg Trp Lys Ala Ala ArgSer His Arg Arg Phe Arg 65 70 75 80 Arg Cys Pro Phe Pro Arg Gln Leu ValTrp Pro Val Arg Glu Arg His 85 90 95 Arg Asp Ala Gly Asp Arg Arg Asn GlnArg Glu Arg Arg Arg Arg Asp 100 105 110 Ala Tyr His Glu Ile Ser Glu ProLys Phe Arg Thr Arg Lys Arg Thr 115 120 125 Glu Ser Phe Trp Met Asn LysAla Ile Thr Val Ile Val Trp Leu Leu 130 135 140 Val Ser Leu Cys Val LeuAla Ile Ile Thr Met Pro Val Ser Leu Gln 145 150 155 160 Thr His Leu ValAla Thr Ala Ile Ser Leu Ile Leu Leu Ala Thr Ile 165 170 175 Lys Ser PheAsn Gly Gln Gly Ala Trp Arg Leu Val Ala Leu Gly Phe 180 185 190 Gly ThrAla Ile Val Leu Arg Tyr Val Tyr Trp Arg Thr Thr Ser Thr 195 200 205 LeuPro Pro Val Asn Gln Leu Glu Asn Phe Ile Pro Gly Phe Leu Leu 210 215 220Tyr Leu Ala Glu Met Tyr Ser Val Val Met Leu Gly Leu Ser Leu Val 225 230235 240 Ile Val Ser Met Pro Leu Pro Ser Arg Lys Thr Arg Pro Gly Ser Pro245 250 255 Asp Tyr Arg Pro Thr Val Asp Val Phe Val Pro Ser Tyr Asn GluAsp 260 265 270 Ala Glu Leu Leu Ala Asn Thr Leu Ala Ala Ala Lys Asn MetAsp Tyr 275 280 285 Pro Ala Asp Arg Phe Thr Val Trp Leu Leu Asp Asp GlyGly Ser Val 290 295 300 Gln Lys Arg Asn Ala Ala Asn Ile Val Glu Ala GlnAla Ala Gln Arg 305 310 315 320 Arg His Glu Glu Leu Lys Lys Leu Cys GluAsp Leu Asp Val Arg Tyr 325 330 335 Leu Thr Arg Glu Arg Asn Val His AlaLys Ala Gly Asn Leu Asn Asn 340 345 350 Gly Leu Ala His Ser Thr Gly GluLeu Val Thr Val Phe Asp Ala Asp 355 360 365 His Ala Pro Ala Arg Asp PheLeu Leu Glu Thr Val Gly Tyr Phe Asp 370 375 380 Glu Asp Pro Arg Leu PheLeu Val Gln Thr Pro His Phe Phe Val Asn 385 390 395 400 Pro Asp Pro IleGlu Arg Asn Leu Arg Thr Phe Glu Thr Met Pro Ser 405 410 415 Glu Asn GluMet Phe Tyr Gly Ile Ile Gln Arg Gly Leu Asp Lys Trp 420 425 430 Asn GlyAla Phe Phe Cys Gly Ser Ala Ala Val Leu Arg Arg Glu Ala 435 440 445 LeuGln Asp Ser Asp Gly Phe Ser Gly Val Ser Ile Thr Glu Asp Cys 450 455 460Glu Thr Ala Leu Ala Leu His Ser Arg Gly Trp Asn Ser Val Tyr Val 465 470475 480 Asp Lys Pro Leu Ile Ala Gly Leu Gln Pro Ala Thr Phe Ala Ser Phe485 490 495 Ile Gly Gln Arg Ser Arg Trp Ala Gln Gly Met Met Gln Ile LeuIle 500 505 510 Phe Arg Gln Pro Leu Phe Lys Arg Gly Leu Ser Phe Thr GlnArg Leu 515 520 525 Cys Tyr Met Ser Ser Thr Leu Phe Trp Leu Phe Pro PhePro Arg Thr 530 535 540 Ile Phe Leu Phe Ala Pro Leu Phe Tyr Leu Phe PheAsp Leu Gln Ile 545 550 555 560 Phe Val Ala Ser Gly Gly Glu Phe Leu AlaTyr Thr Ala Ala Tyr Met 565 570 575 Leu Val Asn Leu Met Met Gln Asn TyrLeu Tyr Gly Ser Phe Arg Trp 580 585 590 Pro Trp Ile Ser Glu Leu Tyr GluTyr Val Gln Thr Val His Leu Leu 595 600 605 Pro Ala Val Val Ser Val IlePhe Asn Pro Gly Lys Pro Thr Phe Lys 610 615 620 Val Thr Ala Lys Asp GluSer Ile Ala Glu Ala Arg Leu Ser Glu Ile 625 630 635 640 Ser Arg Pro PhePhe Val Ile Phe Ala Leu Leu Leu Val Ala Met Ala 645 650 655 Phe Ala ValTrp Arg Ile Tyr Ser Glu Pro Tyr Lys Ala Asp Val Thr 660 665 670 Leu ValVal Gly Gly Trp Asn Leu Leu Asn Leu Ile Phe Ala Gly Cys 675 680 685 AlaLeu Gly Val Val Ser Glu Arg Gly Asp Lys Ser Ala Ser Arg Arg 690 695 700Ile Thr Val Lys Arg Arg Cys Glu Val Gln Leu Gly Gly Ser Asp Thr 705 710715 720 Trp Val Pro Ala Ser Ile Asp Asn Val Ser Val His Gly Leu Leu Ile725 730 735 Asn Ile Phe Asp Ser Ala Thr Asn Ile Glu Lys Gly Ala Thr AlaIle 740 745 750 Val Lys Val Lys Pro His Ser Glu Gly Val Pro Glu Thr MetPro Leu 755 760 765 Asn Val Val Arg Thr Val Arg Gly Glu Gly Phe Val SerIle Gly Cys 770 775 780 Thr Phe Ser Pro Gln Arg Ala Val Asp His Arg LeuIle Ala Asp Leu 785 790 795 800 Ile Phe Ala Asn Ser Glu Gln Trp Ser GluPhe Gln Arg Val Arg Arg 805 810 815 Lys Lys Pro Gly Leu Ile Arg Gly ThrAla Ile Phe Leu Ala Ile Ala 820 825 830 Leu Phe Gln Thr Gln Arg Gly LeuTyr Tyr Leu Val Arg Ala Arg Arg 835 840 845 Pro Ala Pro Lys Ser Ala LysPro Val Gly Ala Val Lys 850 855 860

What is claimed is:
 1. An isolated DNA encoding sequence to a plantcellulose synthesis enzyme.
 2. The DNA encoding sequence of claim 1wherein said cellulose synthesis enzyme is cellulose synthase.
 3. TheDNA encoding sequence of claim 2 wherein said cellulose synthase is fromcotton.
 4. The DNA encoding sequence of claim 3 wherein said cottoncellulose synthase is celA1.
 5. The DNA encoding sequence of claim 4wherein said celA1 is encoded by the sequence of FIG.
 6. 6. The DNAencoding sequence of claim 3 wherein said cotton cellulose synthase iscelA2.
 7. The DNA encoding sequence of claim 6 wherein said celA2 isencoded by the sequence of FIG.
 7. 8. An isolated DNA encoding sequenceto a plant cellulose synthesis promoter region.
 9. The promoter encodingsequence of claim 8 wherein said cellulose synthesis promoter region isto cellulose synthase.
 10. The promoter sequence of claim 9 wherein saidcellulose synthase promoter region is from cotton.
 11. The promotersequence of claim 10 wherein said cotton cellulose synthase promoterregion is from celA1.
 12. The promoter sequence of claim 11 wherein saidcotton cellulose synthase promoter region is the from sequence of FIG.8.
 13. A recombinant DNA construct comprising any of the DNA encodingsequences of claims 1-10.
 14. The DNA construct of claim 13 comprisingas operably joined components in the direction of transcription, acotton fiber transcriptional factor and the sequence of any of claims1-7.
 15. A plant cell comprising a DNA construct of claims 13 or
 14. 16.A plant comprising a cell of claim
 15. 17. A method of modifying fiberphenotype in a cotton plant, said method comprising: transforming aplant cell with DNA comprising a construct of claims 13 or
 14. 18. Amethod of modifying the wood quality phenotype in a forest tree species,said method comprising: transforming a plant cell of said species withDNA comprising a construct of claim
 13. 19. A method according to claim18 wherein said cellulose sythesis enzyme is cellulose synthase andwherein the encoding sequence is in an antisense orientation, whereintranscribed mRNA from said sequence is complementary to the equivalentmRNA transcribed from the endogenous gene, whereby the synthesis ofcellulose in said plant cell is suppressed.
 20. A method according toclaim 18, wherein said cellulose sythesis enzyme is cellulose synthaseand wherein the encoding sequence is in a sense orientation, and whereinthe synthesis of cellulose in said plant cell is increased.
 21. A methodaccording to claim 20 wherein said plant cell additionally comprises aconstruct encoding a sequence to an enzyme involved in the synthesis oflignin or a lignin precursor.
 22. A method according to claim 20 whereinsaid lignin encoding sequence is in an antisense orientation, whereintranscribed mRNA from said sequence is complementary to the equivalentmRNA transcribed from the endogenous gene, whereby the synthesis oflignin is suppressed.